Methods to measure dissociation rates for ligands that form reversible covalent bonds

ABSTRACT

The crystal structure of the ligand binding domain of ERR-α in complex with a ligand that forms a reversible thioether bond to Cys325 of ERR-α, methods to measure dissociation rates for ligands that form reversible covalent bonds, and methods to design ligands that form reversible covalent bonds for use as modulators of ERR-α activity are disclosed. The crystal structure and methods provide a novel molecular mechanism for modulation of the activity of ERR-α and provide the basis for rational drug design to obtain potent specific ligands for use as modulators of the activity of this new drug target.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to application Ser. No. 61/232,983,filed Aug. 11, 2009.

TECHNICAL FIELD

The present invention generally pertains to the fields of proteincrystallization, X-ray diffraction analysis, three-dimensionalstructural determination, molecular modeling, and structure basedrational drug design. The present invention provides a crystallized formof Estrogen Related Receptor alpha (ERR-α) in complex with a ligand thatforms a thioether bond in the ligand binding pocket (LBP), methods tomeasure dissociation rates for ligands that form reversible covalentbonds, and methods to design ligands that form reversible covalent bondsfor use as modulators of ERR-α activity.

BACKGROUND OF THE INVENTION

Various publications, which may include patents, published applications,technical articles and scholarly articles, are cited throughout thespecification in parentheses, and full citations of each may be found atthe end of the specification. Each of these cited publications isincorporated by reference herein, in its entirety.

Nuclear receptors are members of a superfamily of transcription factors.The members of this family share structural similarities and regulate adiverse set of biological effects (Olefsky 2001). Ligands activate orrepress these transcription factors that control genes involved inmetabolism, differentiation and reproduction (Laudet and Gronmeyer2002). Presently, the human genome project has identified about 48members for this family and cognate ligands have been identified forabout 28 of them (Giguere 1999). This protein family is composed ofmodular structural domains that can be interchanged within the membersof the family without loss of function. A typical nuclear receptorcontains a hypervariable N-terminus, a conserved DNA binding domain(DBD), a hinge region, and a conserved ligand binding domain (LBD). Thefunction of the DBD is targeting of the receptor to specific DNAsequences (nuclear hormone response elements or NREs). The function ofthe LBD is recognition of its cognate ligand. Within the sequence of thenuclear receptor there are regions involved in transcriptionalactivation. The AF-1 domain is situated at the N-terminus andconstitutively activates transcription (Rochette-Egly, Gaub et al. 1992;Rochette-Egly, Adam et al. 1997), while the AF-2 domain is embeddedwithin the LBD and its transcriptional activation is ligand dependent(Wurtz, Bourguet et al. 1996). Nuclear receptors can exist as monomers,homodimers or heterodimers and bind to direct or inverted nucleotiderepeats (Aranda and Pascual 2001; Laudet and Gronmeyer 2002).

The members of this family exist either in an activated or repressedbasal biological state. The basic mechanism of gene activation involvesligand dependent exchange of co-regulatory proteins. These co-regulatoryproteins are referred to as co-activators or co-repressors (McKenna,Lanz et al. 1999). A nuclear receptor in the repressed state is bound toits DNA response element and is associated with co-repressor proteinsthat recruit histone de-acetylases (HDACs) (Jones and Shi 2003). In thepresence of an agonist there is an exchange of co-repressors withco-activators that in turn recruit transcription factors that assembleinto an ATP dependent chromatin-remodeling complex. Histones arehyper-acetylated, causing the nucleosome to unfold, and repression isalleviated. The AF-2 domain acts as the ligand dependent molecularswitch for the exchange of co-regulatory proteins. In the presence of anagonist the AF-2 domain undergoes a conformational transition andpresents a surface on the LBD for interaction with co-activatorproteins. In the absence of an agonist or in the presence of anantagonist the AF-2 domain presents a surface that promotes interactionswith co-repressor proteins. The interaction surfaces on the LBD for bothco-activators, and co-repressors overlap and provide a conservedmolecular mechanism for gene activation or repression that is shared bythe members of this family of transcription factors (Xu, Stanley et al.2002).

Natural ligands that modulate the biological activity of nuclearreceptors have been identified for only approximately one half of knownnuclear receptors. Receptors for which no natural ligand has beenidentified are termed “orphan receptors”. The discovery of ligands orcompounds that interact with an orphan receptor will accelerate theunderstanding of the role of the nuclear receptors in physiology anddisease and facilitate the pursuit of new therapeutic approaches. Asub-class of these receptors, for which no natural ligands have beenidentified, is the estrogen related receptors (ERRs).

Estrogen Related Receptor alpha (ERR-α), also known as ERR-1, is anorphan receptor and was the first to be identified of the three membersof the estrogen receptor related subfamily of orphan nuclear receptors(ERR-α, β, γ). The ERR subfamily is closely related to the estrogenreceptors (ER-α and ER-β). ERR-α and ERR-β were first isolated by a lowstringency hybridization screen (Giguere, Yang et al. 1988) followedlater with the discovery of ERR-γ (Hong, Yang et al. 1999). The ERRs andERs share sequence similarity with the highest homology observed intheir DBDs, approximately 60%, and all interact with the classical DNAestrogen response element. Recent biochemical evidence suggested thatthe ERRs and ERs share co-regulator proteins and also target genes,including pS2, lactoferin, aromatase, and osteopontin (Hong, Yang et al.1999; Zhang and Teng 2000; Giguere 2002; Kraus, Ariazi et al. 2002). Ithas been suggested that one of the main functions of ERRs is to regulatethe response of estrogen responsive genes. The effects of the steroidhormone estrogen are primarily mediated in the breast, bone andendometrium, so it is reasonable to believe that compounds that interactwith ERRs may find use for the treatment of bone related disease, breastcancer, and other diseases related to the reproduction system.

For example, it has been shown that ERR-α is present in both normal andcancerous breast tissue (Ariazi, Clark et al. 2002). It has also beenreported that the main function of ERR-α in normal breast tissue is thatof a repressor for estrogen responsive genes. In breast cancers or celllines that are non-estrogen responsive (ER-α negative), ERR-α has beenreported to be in an activated state (Ariazi, Clark et al. 2002).Therefore compounds that interact with ERR-α may be useful agents forthe treatment of breast cancer that is ER-α negative and non-responsiveto classical anti-estrogenic therapy, or may be used as an adjunct agentfor anti-estrogen responsive breast cancers. These agents may act asantagonists by reducing the biological activity of ERR-α in theseparticular tissues.

Regarding bone related diseases, many post-menopausal women experienceosteoporosis, a condition that has been clearly associated with areduction of estrogen production. For example, it has been shown thatreduction of estrogen levels results in increased bone loss (Turner,Riggs et al. 1994). It has also been shown that administration ofestrogens to postmenopausal patients with osteoporosis has an anaboliceffect on bone development (Pacifici 1996). The molecular mechanismlinking estrogen receptors to bone loss is not well understood, however,since ER-α and ER-β knock-out animals have only minor skeletal defects(Korach 1994; Windahl, Vidal et al. 1999). With regard to ERR-α in bone,ERR-α expression has been shown to be regulated by estrogen (Bonnelye,Vanacker et al. 1997; Bonnelye, Merdad et al. 2001) and ERR-α expressionis known to be maintained throughout stages of osteoblastdifferentiation. Furthermore, over-expression of ERR-α in rat calvariaosteoblasts, an accepted model of bone differentiation, resulted in anincrease of bone nodule formation and treatment of rat calvariaosteoblasts with ERR-α antisense results in a decrease of bone noduleformation. ERR-α also regulates osteopontin, a protein believed to beinvolved in bone matrix formation. Therefore, compounds that modulateERR-α by increasing its activity may have an anabolic effect for theregeneration of bone density and provide a benefit over currentapproaches that prevent bone loss. Such compounds may enhance theactivity of the receptor by enhancing the association of the receptorwith proteins that increase its activity or improve the stability of thereceptor or by increasing the intracellular concentrations of thereceptor and consequently increasing its activity. Conversely, withrespect to bone diseases that are a result of abnormal bone growth,compounds that interact with ERR-α and decrease its biological activitymay provide a benefit for the treatment of these diseases by retardingbone growth. Antagonism of the association of the receptor withco-activator proteins decreases the activity of the receptor.

ERR-α is also present in cardiac, adipose, and muscle tissue and forms atranscriptionally active complex with the PGC-1 co-activator family,which are co-activators implicated in energy homeostasis, mitochondriabiogenesis, hepatic gluconeogenesis and in the regulation of genesinvolved in fatty acid beta-oxidation (Kamei, Ohizumi et al. 2003).ERR-α regulates the expression of medium chain acyl-CoA dehydrogenase(MCAD) through interactions with its promoter. MCAD is a gene involvedin the initial reaction in fatty acid beta-oxidation. It is believedthat in the adipose tissue, ERR-α regulates energy expenditure throughthe regulation of MCAD (Sladek, Bader et al. 1997; Vega and Kelly 1997).In antisense experiments in rat calvaria osteoblasts, in addition to theinhibition of bone nodule formation, there was an increase in adipocytedifferentiation markers including aP2 and PPAR-T (Bonnelye, Kung et al.2002). An ERR-α knockout model has been described that exhibited reducedfat mass relative to the wild type. DNA chip analysis indicated that theERR-α knockout mice have an alteration in the expression levels of genesinvolved in adipogenesis and energy metabolism (Luo, Sladek et al.2003). More recently it has been shown that ERR-α regulates theexpression of endothelial nitric oxide synthase, a gene that has aprotective mechanism against arteriosclerosis (Sumi and Ignarro 2003).The biochemical evidence supports the involvement of ERR-α in metabolichomeostasis and differentiation of cells into adipocytes. Therefore,compounds interacting with ERR-α may affect energy homeostasis andprovide a benefit for the treatment of obesity and metabolic syndromerelated disease indications, including arteriosclerosis and diabetes(Grundy, Brewer et al. 2004).

Lion Bioscience AG disclosed the use of certain pyrazole derivatives asantagonists of ERR-α for treating cancer, osteoporosis, obesity, lipiddisorders and cardiovascular disorders and for regulating fertility(US20060148876). Still other small molecules were also disclosed asERR-α modulators (US20060014812; US20080221179).

There is a continuing need for new ERR-α inverse agonists that may finduse in the treatment of conditions including but not limited tobone-related disease, bone formation, breast cancer (including thoseunresponsive to anti-estrogen therapy), cartilage formation, cartilageinjury, cartilage loss, cartilage degeneration, cartilage injury,ankylosing spondylitis, chronic back injury, gout, osteoporosis,osteolytic bone metastasis, multiple myeloma, chondrosarcoma,chondrodysplasia, osteogenesis imperfecta, osteomalacia, Paget'sdisease, polymyalgia rheumatica, pseudogout, arthritis, rheumatoidarthritis, infectious arthritis, osteoarthritis, psoriatic arthritis,reactive arthritis, childhood arthritis, Reiter's syndrome, repetitivestress injury, periodontal disease, chronic inflammatory airway disease,chronic bronchitis, chronic obstructive pulmonary disease, metabolicsyndrome, obesity, disorders of energy homeostasis, diabetes, lipiddisorders, cardiovascular disorders, artherosclerosis, hyperglycemia,elevated blood glucose level, and insulin resistance.

X-ray crystal structures provide powerful tools for the rational designof ligands that can function as active agents for biologically importanttargets. The first crystal structure solved for ERR-α was a complex ofthe ERR-α ligand binding domain and a coactivator peptide fromperoxisome proliferator-activated receptor coactivator-1 (PGC-1)(Kallen, Schlaeppi et al. 2004). The structure revealed that theputative ligand binding pocket (LBP) of ERR-α is almost completelyoccupied by side chains, in particular with the bulky side chain ofPhe328. The crystal structure of ERR-α in a transcriptionally activeconformation, in the absence of a ligand, provided evidence forligand-independent transcriptional activation by ERR-α. A second ERR-αcrystal structure was solved with the ligand binding domain of ERR-α(containing a C325S mutation) in complex with an inverse agonist boundin the ligand binding pocket (LBP). The C325S mutation was introduced toreduce biochemical instability problems during purification andcrystallization that were determined to be associated with cysteineoxidation. (Kallen, Lattmann et al. 2007). The structure revealed adramatic conformational change in the ERR-α LBP which created thenecessary space for the ligand to bind. Due to the C325S mutation in theLBP, however, the structure left unresolved the importance of the Cys325in designing ligands for use as modulators of ERR-α activity.

It has been shown that certain ligands form a covalent bond to acysteine residue in the peroxisome proliferator-activated receptor(PPAR) ligand binding domain through a Michael addition, and thatcovalent binding is required for PPAR activation by the ligands(Shiraki, Kamiya et al. 2005). Covalent binding has also beendemonstrated in a number of different drugs for a variety of drugtargets. A few examples are briefly included below. It was proposed thattargeted covalent inactivation of a variety of protein kinases may holdpromise for developing treatments for a number of different diseases(US20060079494; Fry, Bridges et al. 1998; Schirmer, Kennedy et al. 2006;Wood, Shewchuk et al. 2008). Covalent binding was also demonstrated forpotent and species-specific inhibitors of 3-hydroxy-3-methylglutaryl CoAsynthases ((Pojer, Ferrer et al. 2006). It was shown that F-amidine andCl-amidine irreversibly inactivate protein arginine deiminase 4 (PAD4)in a calcium-dependent manner via the specific modification of Cys645,an active site residue that is critical for catalysis. A growing body ofevidence supports a role for PAD4 in the onset and progression ofrheumatoid arthritis, a chronic autoimmune disorder. It was concludedthat the covalent binding compounds may be useful as potential leadcompounds for the treatment of rheumatoid arthritis (Luo, Arita et al.2006). Even the unique properties of aspirin, the ubiquitousnonsteroidal anti-inflammatory drug, derive from its ability tocovalently modify cyclooxygenases, COX-1 and COX-2, the in vivo targetsfor its action (Kalgutkar, Crews et al. 1998).

The present invention provides a crystallized form of a complex of theERR-α ligand binding domain (ERR-α-LBD) with a ligand that forms athioether bond to Cys325 of ERR-α. The diffraction pattern of thecrystal is of sufficient resolution so that the three-dimensionalstructure of ERR-α can be determined at atomic resolution,ligand-binding sites on ERR-α can be identified, and the interactions ofligands with specific amino acid residues of ERR-α can be modeled andused to design ligands that can function as active agents. The assaymethods of the present invention can be used to measure dissociationrates for ligands that form reversible covalent bonds and can functionas active agents. Thus, the three-dimensional structure of the complexof the ERR-α ligand binding domain (ERR-α-LBD) with a ligand that formsa thioether bond to Cys325 and the assay methods of the presentinvention have applications to the design and biologicalcharacterization of ligands that function as modulators of ERR-αactivity. Such ligands may be useful for treating, ameliorating,preventing or inhibiting the progression of disease states, disordersand conditions that are mediated by ERR-α activity.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda method of producing and using three-dimensional structure informationderived from the crystal structure of a complex of Estrogen RelatedReceptor alpha (ERR-α) and Compound 1.

The present invention also includes specific crystallization conditionsto obtain crystals of the complex of ERR-α and Compound 1. The crystalsare subsequently used to obtain a 3-dimensional structure of the complexusing X-ray crystallography. The obtained data is used for rational drugdiscovery with the aim to design ligands that are modulators of ERR-αactivity.

The present invention includes a crystal comprising ERR-α, or afragment, or target structural motif or derivative thereof, and aligand, wherein the ligand forms a thioether bond to Cys325 of ERR-α.

In another embodiment, the present invention comprises a crystal ofERR-α and a ligand, wherein said ligand forms a thioether bond to Cys325of ERR-α, the crystal having a spacegroup of P6522.

In yet another embodiment, the present invention comprises a crystal ofERR-α and a ligand, wherein said ligand forms a thioether bond to Cys325of ERR-α, the crystal with a unit cell having dimensions of abouta=b=103.007 and c=110.017.

In another aspect of the invention, the invention includes a computersystem comprising: (a) a database stored on a computer readable storagemedium, the database containing information on the three dimensionalstructure of a crystal comprising ERR-α, or a fragment or a targetstructural motif or derivative thereof, and a ligand, wherein saidligand that forms a thioether bond to Cys325 of ERR-α; and, (b) a userinterface to view the information.

Further included in the present invention is a method of identifying amodulator of ERR-α activity, comprising: (a) employing the threedimensional structure of ERR-α cocrystallized with a ligand that forms athioether bond to Cys325 of ERR-α; and, (b) designing or selecting saidmodulator of ERR-α activity, thereby identifying the modulator of ERR-αactivity.

The invention comprises a method of locating the attachment site of amodulator of ERR-α activity, comprising: (a.) contacting human ERR-αwith the ligand; (b.) cocrystallizing human ERR-α with the ligand; (c.)obtaining X-ray diffraction data for a complex of ERR-α and the ligand;(d.) obtaining X-ray diffraction data for a crystal of ERR-α without theligand; (e.) subtracting the X-ray diffraction data obtained in step (c)from the X-ray diffraction data obtained in step (d) to obtain thedifference in the X-ray diffraction data; (f.) obtaining phases thatcorrespond to X-ray diffraction data obtained in step (c); (g.)utilizing the phases obtained in step (f) and the difference in theX-ray diffraction data obtained in step (e) to compute a differenceFourier image of the ligand; and, (h.) locating the attachment site ofthe ligand to ERR-α based on the computations obtained in step (g).

The invention additionally comprises a method for solving the crystalstructure of a complex comprising an ERR-α polypeptide and a ligand thatforms a thioether bond with Cys325 of ERR-α, comprising: (a) contactingthe ERR-α polypeptide with said ligand in a suitable solution comprisingammonium sulfate, Pipes pH 6.5 and Na-thiocyanate; (b) crystallizingsaid resulting complex of the ERR-α polypeptide and the ligand from saidsolution; and, (c) solving the crystal structure of the complex of theERR-α polypeptide and the ligand.

The invention includes a method for identifying a potential modulator ofERR-α activity, comprising: (a) using a three dimensional structure ofthe complex of ERR-α and a ligand that forms a thioether bond withCys325 of ERR-α as defined by atomic coordinates according to Table 6;(b) replacing one or more ERR-α amino acids in said three-dimensionalstructure with a different amino acid to produce a modified ERR-α; (c)using said three-dimensional structure to design or select saidpotential modulator of ERR-α activity; (d) synthesizing said potentialmodulator; and, (e) contacting said potential modulator with saidmodified ERR-α and determining the ability of said potential modulatorto affect said ERR-α activity. Also included in the invention is amodulator of ERR-α identified by the method.

In another embodiment, the present invention provides a method tomeasure the dissociation rate for a ligand that forms a reversiblecovalent bond with a protein, comprising the steps of: (a) measuring byLC/MS a mass for the protein, a mass for a first ligand that forms areversible covalent bond with the protein, and a mass for a competingsecond ligand that forms a reversible covalent bond with the protein,wherein the competing second ligand has a different mass than the firstligand; (b) mixing the protein and the first ligand in a solution withthe first ligand in molar excess of the protein; (c) incubating theprotein and the first ligand in the solution to allow for aprotein:first ligand complex to form; (d) removing an aliquot of thesolution and measuring by LC/MS the mass for the protein:first ligandcomplex; (e) adding molar excess of the competing second ligand to thesolution containing the protein:first ligand complex; (f) removingaliquots of the solution at time 0 and at regular intervals; (g)measuring the time-dependent change in the mass of the protein:firstligand complex; and, (h) determining the dissociation rate for the firstligand; thereby measuring the dissociation rate of a ligand that forms areversible covalent bond with a protein.

The present invention further provides a method to measure thedissociation rate for a ligand that forms a reversible covalent bondwith a protein, wherein the reversible covalent bond is a thioether bondto a cysteine (Cys).

The present invention also provides a method to measure the dissociationrate for a ligand that forms a reversible covalent bond with a protein,wherein the protein comprises Estrogen Related Receptor alpha (ERR-α)and the ligand that forms a thioether bond to Cys325 of ERR-α.

In its many embodiments, the present invention provides methods toidentify novel ligands that may find use as modulators of ERR-αactivity, for example, ligands that function as inverse agonists ofERR-α and form a thioether bond with Cys325 of ERR-α, pharmaceuticalcompositions comprising one or more such ligands, methods of preparingpharmaceutical compositions comprising one or more such ligands, andmethods of treatment, prevention, inhibition or amelioration of one ormore diseases associated with ERR-α activity using such ligands orpharmaceutical compositions containing such ligands.

Another aspect of the present invention features a pharmaceuticalcomposition comprising at least one ligand that forms a thioether bondwith Cys325 of ERR-α and at least one pharmaceutically acceptablecarrier.

The present invention also features a method of treating a subjectsuffering from or diagnosed with a disease, disorder, or conditionmediated by ERR-α activity, comprising administering to the subject atherapeutically effective amount of at least one ligand that forms athioether bond with Cys325 of ERR-α. Such disease, disorder, orcondition can include bone-related disease, bone formation, breastcancer (including those unresponsive to anti-estrogen therapy),cartilage formation, cartilage injury, cartilage loss, cartilagedegeneration, cartilage injury, ankylosing spondylitis, chronic backinjury, gout, osteoporosis, osteolytic bone metastasis, multiplemyeloma, chondrosarcoma, chondrodysplasia, osteogenesis imperfecta,osteomalacia, Paget's disease, polymyalgia rheumatica, pseudogout,arthritis, rheumatoid arthritis, infectious arthritis, osteoarthritis,psoriatic arthritis, reactive arthritis, childhood arthritis, Reiter'ssyndrome, repetitive stress injury, periodontal disease, chronicinflammatory airway disease, chronic bronchitis, chronic obstructivepulmonary disease, metabolic syndrome, obesity, disorders of energyhomeostasis, diabetes, lipid disorders, cardiovascular disorders,artherosclerosis, hyperglycemia, elevated blood glucose level, andinsulin resistance. The therapeutically effective amount of the ligandthat forms a thioether bond with Cys325 of ERR-α can be from about 0.1mg/day to about 5000 mg/day for an average human.

The present invention further includes a method of treating a subjectsuffering from or diagnosed with a disease, disorder, or medicalcondition mediated by Estrogen Related Receptor alpha (ERR-α) activity,comprising administering to the subject an effective amount to treat thedisease, disorder, or medical condition, a ligand that forms a thioetherbond to Cys325 of ERR-α, or a pharmaceutically acceptable salt,pharmaceutically acceptable prodrug, or pharmaceutically activemetabolite thereof, wherein the disease, disorder, or medical conditionis selected from the group consisting of: bone-related disease, boneformation, cartilage formation, cartilage loss, cartilage degeneration,cartilage injury, ankylosing spondylitis, chronic back injury, gout,osteoporosis, osteolytic bone metastasis, multiple myeloma,chondrosarcoma, chondrodysplasia, osteogenesis imperfecta, osteomalacia,Paget's disease, polymyalgia rheumatica, pseudogout, arthritis,rheumatoid arthritis, infectious arthritis, osteoarthritis, psoriaticarthritis, reactive arthritis, childhood arthritis, Reiter's syndrome,repetitive stress injury, periodontal disease, chronic inflammatoryairway disease, chronic bronchitis, chronic obstructive pulmonarydisease, breast cancer, metabolic syndrome, obesity, disorders of energyhomeostasis, diabetes, lipid disorders, cardiovascular disorders, andartherosclerosis.

The present invention provides a method of treating a subject sufferingfrom or diagnosed with a disease, disorder, or medical conditionmediated by ERR-α activity, comprising administering to the subject apharmaceutical composition comprising: (a) an effective amount of apharmaceutical agent to treat the disease, disorder, or medicalcondition, said pharmaceutical agent comprising a ligand that forms athioether bond to Cys325 of ERR-α and pharmaceutically acceptable salts,pharmaceutically acceptable prodrugs, and pharmaceutically activemetabolites of said compounds; and, (b) a pharmaceutically acceptableexcipient, wherein the disease, disorder, or medical condition isbone-related disease, bone formation, cartilage formation, cartilageloss, cartilage degeneration, cartilage injury, ankylosing spondylitis,chronic back injury, gout, osteoporosis, osteolytic bone metastasis,multiple myeloma, chondrosarcoma, chondrodysplasia, osteogenesisimperfecta, osteomalacia, Paget's disease, polymyalgia rheumatica,pseudogout, arthritis, rheumatoid arthritis, infectious arthritis,osteoarthritis, psoriatic arthritis, reactive arthritis, childhoodarthritis, Reiter's syndrome, repetitive stress injury, periodontaldisease, chronic inflammatory airway disease, chronic bronchitis,chronic obstructive pulmonary disease, breast cancer, metabolicsyndrome, obesity, energy disorder, homeostasis, diabetes, lipiddisorder, cardiovascular disorder, or artherosclerosis.

Additional embodiments and advantages of the invention will becomeapparent from the detailed discussion, schemes, examples, and claimsbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

A preferred embodiment of the present invention will now be described,by way of an example only, with reference to the accompanying drawingswherein:

FIG. 1: A. Shown is a ribbon representation of an overlay of the complexof ERR-α and PGC1-α onto the complex of ERR-α and Compound 1. The ERR-αprotein of the complex of ERR-α and Compound 1 is depicted in green withCompound 1 depicted in cyan. The complex of ERR-α and PGC1-α is shown inmagenta with the PGC1-α peptide in red. B. Shown is a stick modelrepresentation of an overlay of complex of ERR-α and PGC1-α onto thecomplex of ERR-α and Compound 1.

FIG. 2: A. Shown is a 2fofc map calculated to 1.6σ around Compound 1. B.Shown is a sulfur anomalous map calculated to 3.5σ, that validates thecorrect orientation of the thiazole of Compound 1 and Cys325 of ERR-α.

DEFINITIONS

As is generally the case in biotechnology and chemistry, the descriptionof the present invention has required the use of a number of terms ofart. Although it is not practical to do so exhaustively, definitions forsome of these terms are provided here for ease of reference. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Definitions for other terms mayalso appear elsewhere herein. However, the definitions provided here andelsewhere herein should always be considered in determining the intendedscope and meaning of the defined terms. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice of the present invention, the preferred methods andmaterials are described.

The term “comprising” means “including principally, but not necessarilysolely”. Furthermore, variations of the word “comprising”, such as“comprise” and “comprises”, have correspondingly varied meanings.

As used herein, the terms “containing”, “having” and “including” areused in their open, non-limiting sense.

As used herein, “sequence” means the linear order in which monomersoccur in a polymer, for example, the order of amino acids in apolypeptide or the order of nucleotides in a polynucleotide.

The terms “polypeptide”, “protein”, and “peptide” are used hereininterchangeably to refer to amino acid chains in which the amino acidresidues are linked by peptide bonds or modified peptide bonds. Theamino acid chains can be of any length of greater than two amino acids.Unless otherwise specified, the terms “polypeptide”, “protein”, and“peptide” also encompass various modified forms thereof. Such modifiedforms may be naturally occurring modified forms or chemically modifiedforms. Examples of modified forms include, but are not limited to,glycosylated forms, phosphorylated forms, myristoylated forms,palmitoylated forms, ribosylated forms, acetylated forms, ubiquitinatedforms, etc. Modifications also include intra-molecular crosslinking andcovalent attachment to various moieties such as lipids, flavin, biotin,polyethylene glycol or derivatives thereof, etc. In addition,modifications may also include cyclization, branching and cross-linking.Further, amino acids other than the conventional twenty amino acidsencoded by the codons of genes may also be included in a polypeptide.

As used herein, a protein or nucleic acid molecule is said to be“isolated” when the protein or nucleic acid molecule is substantiallyseparated from contaminants from the source of the protein or nucleicacid.

As used herein, the term “native protein” refers to a protein comprisingan amino acid sequence identical to that of a protein isolated from itsnatural source or organism.

As used herein, the term “amino acids” refers to the L-isomers of thenaturally occurring amino acids. The naturally occurring amino acids areglycine, alanine, valine, leucine, isoleucine, serine, methionine,threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline,histidine, aspartic acid, asparagine, glutamic acid, glutamine,γ-carboxylglutamic acid, arginine, ornithine, and lysine. Unlessspecifically indicated, all amino acids are referred to in thisapplication are in the L-form.

As used herein, the term “nonnatural amino acids” refers to amino acidsthat are not naturally found in proteins. For example, selenomethionine.

As used herein, the term “positively charged amino acid” includes anyamino acids having a positively charged side chain under normalphysiological conditions. Examples of positively charged naturallyoccurring amino acids are arginine, lysine, and histidine.

As used herein, the term “negatively charged amino acid” includes anyamino acids having a negatively charged side chains under normalphysiological conditions. Examples of negatively charged naturallyoccurring amino acids are aspartic acid and glutamic acid.

As used herein, the term “hydrophobic amino acid” includes any aminoacids having an uncharged, nonpolar side chain that is relativelyinsoluble in water. Examples of naturally occurring hydrophobic aminoacids are alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine.

As used herein, the term “hydrophilic amino acid” refers to any aminoacids having an uncharged, polar side chain that is relatively solublein water. Examples of naturally occurring hydrophilic amino acids areserine, threonine, tyrosine, asparagine, glutamine and cysteine.

As used herein, “nucleic acid” is defined as RNA or DNA that encodes aprotein or peptide as defined herein, or is complementary to nucleicacid sequence encoding such peptides, or hybridizes to such nucleic acidand remains stably bound to it under appropriate stringency conditions.Nucleic acid sequences can be composed of natural nucleotides of thefollowing bases: thymidine, adenine, cytosine, guanine, and uracil;abbreviated T, A, C, G, and U, respectively, and/or synthetic analogs ofthe natural nucleotides.

The term “oligonucleotide” or “oligo” refers to a single-stranded DNA orRNA sequence of a relatively short length, for example, less than 100residues long. For many methods, oligonucleotides of about 16-25nucleotides in length are useful, although longer oligonucleotides ofgreater than about 25 nucleotides may sometimes be utilized. Someoligonucleotides can be used as “primers” for the synthesis ofcomplimentary nucleic acid strands. For example, DNA primers canhybridize to a complimentary nucleic acid sequence to prime thesynthesis of a complimentary DNA strand in reactions using DNApolymerases. Oligonucleotides are also useful for hybridization inseveral methods of nucleic acid detection, for example, in Northernblotting or in situ hybridization.

“Recombinant” refers to a nucleic acid, a protein encoded by a nucleicacid, a cell, or a viral particle, that has been modified usingmolecular biology techniques to something other than its natural state.For example, recombinant cells can contain nucleotide sequence that isnot found within the native (non-recombinant) form of the cell or canexpress native genes that are otherwise abnormally, under-expressed, ornot expressed at all. Recombinant cells can also contain genes found inthe native form of the cell wherein the genes are modified andre-introduced into the cell by artificial means. The term alsoencompasses cells that contain an endogenous nucleic acid that has beenmodified without removing the nucleic acid from the cell; suchmodifications include those obtained, for example, by gene replacement,and site-specific mutation.

The term “high stringency” as used herein refers to the conditions underwhich two nucleic acids may be hybridized, and may include, for example,the concentration of salts and/or detergents in a solution, thetemperature of a solution that is used during the hybridization of thetwo nucleic acids and time period of the hybridization. Accordingly, theterm “high stringency” as used herein refers to conditions in a solutionthat are conducive to hybridization of two nucleic acids only where suchnucleic acids share a high degree of complementarity. The degree ofcomplementarity may include, but not be limited to, a range of fromabout 90% to 100%. Thus, “high stringency” conditions may involve, butare not limited to, the use of a varying temperature and a buffercomprising various concentrations of detergents, salts, and divalentcations.

As used herein, “vector” refers to a nucleic acid molecule into which aheterologous nucleic acid can be or is inserted. Some vectors can beintroduced into a host cell allowing for replication of the vector orfor expression of a protein that is encoded by the vector or construct.Vectors typically have selectable markers, for example, genes thatencode proteins allowing for drug resistance, origins of replicationsequences, and multiple cloning sites that allow for insertion of aheterologous sequence. Vectors are typically plasmid-based and aredesignated by a lower case “p” followed by a combination of lettersand/or numbers. Starting plasmids disclosed herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids by application of proceduresknown in the art. Many plasmids and other cloning and expression vectorsthat can be used in accordance with the present invention are well-knownand readily available to those of skill in the art. Moreover, those ofskill readily may construct any number of other plasmids suitable foruse in the invention. The properties, construction and use of suchplasmids, as well as other vectors, in the present invention will bereadily apparent to those of skill from the present disclosure.

As used herein, the term “activity” refers to an activity exerted byERR-α as determined in vivo or in vitro, according to standardtechniques. Examples of such activity include, but are not limited to,direct activity such as the ability to bind to a ligand or an analogthereof, changes in transcriptional activity, changes in the levels ofgenes or gene products that are regulated directly or indirectly byERR-α activity, changes in enzymatic activity for protein whoseexpression may be affected directly or indirectly by ERR-α activity, orfunctional changes of cell physiology that result from changes in ERR-αactivity.

The term “high-throughput assay” or “high-throughput screening” refersto assay designs that allow easy screening of multiple samplessimultaneously and/or in rapid succession, and may include the capacityfor robotic manipulation. Another desired feature of high-throughputassays is an assay design that is optimized to reduce reagent usage, orminimize the number of manipulations in order to achieve the analysisdesired. Examples of high-throughput assay formats include, but are notlimited to, formats that utilize 96-well, 384-well, and 1536-wellplates, or “lab on a chip” microchannel chips used for liquid handlingexperiments. It is well known by those in the art that asminiaturization of plastic molds and liquid handling devices areadvanced, or as improved assay devices are designed, greater numbers ofsamples can be processed using the forms of the present invention. Anyhigh-throughput screening may be utilized to test new compounds, whichare identified or designed for their ability to interact with ERR-α. Forgeneral information on high-throughput screening see, for example,(Devlin (editor) 1998); and U.S. Pat. No. 5,763,263.

By the term “selecting” or “select” compounds it is intended toencompass both (a) choosing compounds from a group previously unknown tobe modulators of a protein complex or interacting protein membersthereof; and (b) testing compounds that are known to be capable ofbinding, or modulating the functions and activities of, a proteincomplex or interacting protein members thereof. The compounds encompassnumerous chemical classes, including but not limited to, small organicor inorganic compounds, natural or synthetic molecules, such asantibodies, proteins or fragments thereof, antisense nucleotides,interfering RNA (iRNA) and ribozymes, and derivatives, mimetics andanalogs thereof. Preferably, they are small organic compounds, i.e.,those having a molecular weight of no greater than 10,000 daltons, morepreferably less than 5,000 daltons.

As used herein, the term “atomic coordinates” or “structure coordinates”refers to mathematical coordinates that describe the positions of atomsin crystals of ERR-α in Protein Data Bank (PDB) format, including X, Y,Z and B, for each atom. The diffraction data obtained from the crystalsare used to calculate an electron density map of the repeating unit ofthe crystal. The electron density maps may be used to establish thepositions (i.e. coordinates X, Y and Z) of the individual atoms withinthe crystal. Those of skill in the art understand that a set ofstructure coordinates determined by X-ray crystallography is not withoutstandard error. For the purpose of this invention, any set of structurecoordinates for a complex of ERR-α and a ligand that forms a thioetherbond to Cys325 of ERR-α from any source having a root mean squaredeviation of non-hydrogen atoms of less than about 1.5 Å whensuperimposed on the non-hydrogen atom positions of the correspondingatomic coordinates of Table 6 are considered substantially identical orhomologous. In a more preferred embodiment, any set of structurecoordinates for a complex of ERR-α and a ligand that forms a thioetherbond to Cys325 of ERR-α from any source having a root mean squaredeviation of non-hydrogen atoms of less than about 0.75 Å whensuperimposed on the non-hydrogen atom positions of the correspondingatomic coordinates of Table 6 are considered substantially identical orhomologous.

The term “atom type” refers to the chemical element whose coordinatesare measured. The abbreviations in column 3 of Table 6 identifies theelement.

The terms “X,” “Y” and “Z” refer to the crystallographically-definedatomic position of the element measured with respect to the chosencrystallographic origin. The term “B” refers to a thermal factor thatmeasures the mean variation of an atom's position with respect to itsaverage position.

As used herein, the term “crystal” refers to any three-dimensionalordered array of molecules that diffracts X-rays.

As used herein, the term “carrier” in a composition refers to a diluent,adjuvant, excipient, or vehicle with which the product is mixed.

As used herein, the term “composition” refers to the combining ofdistinct elements or ingredients to form a whole. A compositioncomprises more than one element or ingredient. For the purposes of thisinvention, a composition will often, but not always comprise a carrier.

As used herein, “ERR-α” is used to mean a protein obtained as a resultof expression of human Estrogen Related Receptor alpha. Within themeaning of this term, it will be understood that human ERR-α encompassesall proteins encoded by Estrogen Related Receptor alpha, mutantsthereof, conservative amino acid substitutions, alternative spliceproteins thereof, and phosphorylated proteins thereof. Additionally, asused herein, it will be understood that the term “ERR-α” includes humanEstrogen Related Receptor alpha and homologues from other animals. As anexample, ERR-α includes the protein comprising SEQ ID NO:1 and variantsthereof comprising at least about 70% amino acid sequence identity toSEQ ID NO:1, or preferably 80%, 85%, 90% and 95% sequence identity toSEQ ID NO:1, or more preferably, at least about 95% or more sequenceidentity to SEQ ID NO:1.

As used herein, the term “SAR”, an abbreviation for Structure-ActivityRelationships, collectively refers to the structure-activity/structureproperty relationships pertaining to the relationship(s) between acompound's activity/properties and its chemical structure.

As used herein, the term “molecular structure” refers to the threedimensional arrangement of molecules of a particular compound or complexof molecules (e.g., the three dimensional structure of a complex ofERR-α and a that ligand that forms a thioether bond to Cys325 of ERR-α).

As used herein, the term “molecular modeling” refers to the use ofcomputational methods, preferably computer assisted methods, to drawrealistic models of what molecules look like and to make predictionsabout structure activity relationships of ligands. The methods used inmolecular modeling range from molecular graphics to computationalchemistry.

As used herein, the term “molecular model” refers to the threedimensional arrangement of the atoms of a molecule connected by covalentbonds or the three dimensional arrangement of the atoms of a complexcomprising more than one molecule, e.g., a protein:ligand complex.

As used herein, the term “molecular graphics” refers to threedimensional (3D) representations of the molecules; for instance, a 3Drepresentation produced using computer assisted computational methods.

As used herein, “computer readable medium” refers to any medium, whichcan be read and accessed directly by a computer. Such media include, butare not limited to: magnetic storage media, such as floppy discs, harddisc storage media, and magnetic tape; optical storage media such asoptical discs or CD-ROM; electrical storage media such as RAM and ROM;and hybrids of these categories such as magnetic/optical storage media.

As used herein, “recorded” refers to a process for storing informationon computer readable media. A skilled artisan can readily adopt any ofthe presently known methods for recording information on computerreadable media to generate compositions comprising an amino acidsequence and/or atomic coordinate/X-ray diffraction data information ofthe present invention.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the sequenceand/or X-ray diffraction data of the present invention. The minimumhardware means of the computer-based systems of the present inventioncomprises a central processing unit (CPU), input means, output means,and data storage means. A skilled artisan can readily appreciate whichof the currently available computer-based systems are suitable for usein the present invention. A visualization device, such as a monitor, isoptionally provided to visualize structure data.

As stated above, the computer-based systems of the present inventioncomprise a data storage means having stored therein sequence and/oratomic coordinate/X-ray diffraction data of the present invention andthe necessary hardware means and software means for supporting andimplementing an analysis means. As used herein, “data storage means”refers to memory which can store sequence or atomic coordinate/X-raydiffraction data of the present invention, or a memory access meanswhich can access manufactures having recorded thereon the sequence orX-ray data of the present invention.

As used herein, “search means” or “analysis means” refers to one or moreprograms which are implemented on the computer-based system to compare atarget sequence or target structural motif with the sequence or X-raydata stored within the data storage means. Search means are used toidentify fragments or regions of a protein which match a particulartarget sequence or target motif. A variety of known algorithms aredisclosed publicly and a variety of commercially available software forconducting search means are and can be used in the computer-basedsystems of the present invention. A skilled artisan can readilyrecognize that any one of the available algorithms or implementingsoftware packages for conducting computer analyses can be adapted foruse in the present computer-based systems.

As used herein, “a target structural motif”, or “target motif”, refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configuration orelectron density map which is formed upon the folding of the targetmotif. There are a variety of target motifs known in the art. Proteintarget motifs include, but are not limited to, enzymatic active sites,inhibitor binding sites, structural subdomains, epitopes, functionaldomains and signal sequences. Similar motifs are known for RNA. Avariety of structural formats for the input and output means can be usedto input and output the information in the computer-based systems of thepresent invention.

As used herein, the term “computational chemistry” refers tocalculations of the physical and chemical properties of the molecules.

As used herein, the term “molecular replacement” refers to a method thatinvolves generating a preliminary model of a crystal of a complex ofERR-α and a ligand that forms a thioether bond to Cys325 of ERR-α whosecoordinates are unknown, by orienting and positioning the atomiccoordinates described in the present invention so as best to account forthe observed diffraction pattern of the unknown crystal. Phases can thenbe calculated from this model and combined with the observed amplitudesto give an approximate Fourier synthesis of the structure whosecoordinates are unknown. (Rossmann 1972).

As used herein, the term “homolog” refers to the ERR-α protein moleculeor the nucleic acid molecule which encodes the protein, or a functionaldomain from said protein from a first source having at least about 70%or 75% sequence identity, or at least about 80% sequence identity, ormore preferably at least about 85% sequence identity, or even morepreferably at least about 90% sequence identity, and most preferably atleast about 95%, 97% or 99% sequence identity, with the amino acidsequence of the protein, the encoding nucleic acid molecule, or anyfunctional domain thereof, from a second source. The second source maybe a version of the molecule from the first source that has beengenetically altered by any available means to change the primary aminoacid or nucleotide sequence or may be from the same or a differentspecies than that of the first source.

As used herein, the term “active site” refers to regions on ERR-α or astructural motif of ERR-α that are directly involved in the function oractivity of human ERR-α.

As used herein, the terms “binding site” or “binding pocket” refer to aregion of human ERR-α or a molecular complex comprising ERR-α that, as aresult of the primary amino acid sequence of human ERR-α and/or itsthree-dimensional shape, favourably associates with another chemicalentity or compound including ligands, cofactors, inhibitors, or othertypes of modulators. For the purpose of this invention, any active site,binding site or binding pocket defined by a set of structure coordinatesfor a complex of ERR-α or a homolog of ERR-α and a ligand that forms athioether bond to Cys325 of ERR-α or a homolog of ERR-α from any sourcehaving a root mean square deviation of non-hydrogen atoms of less thanabout 1.5 Å when superimposed on the non-hydrogen atom positions of thecorresponding atomic coordinates of Table 6 are considered substantiallyidentical or homologous. In a more preferred embodiment, any set ofstructure coordinates for a complex of ERR-α or a homolog of ERR-α and aligand that forms a thioether bond to Cys325 of ERR-α or a homolog ofERR-α from any source having a root mean square deviation ofnon-hydrogen atoms of less than about 0.75 Å when superimposed on thenon-hydrogen atom positions of the corresponding atomic coordinates ofTable 6 are considered substantially identical or homologous.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean.

As used herein, the term “hydrogen bond” refers to two hydrophilic atoms(either O or N), which share a hydrogen that is covalently bonded toonly one atom, while interacting with the other.

As used herein, the term “hydrophobic interaction” refers tointeractions made by two hydrophobic residues or atoms (such as carbon).

As used herein, the term “conjugated system” refers to more than twodouble bonds adjacent to each other, in which electrons are completelydelocalized with the entire system. This also includes aromaticresidues.

As used herein, the term “aromatic residue” refers to amino acids withside chains having a delocalized conjugated system. Examples of aromaticresidues are phenylalanine, tryptophan, and tyrosine.

As used herein, the phrase “inhibiting the binding” refers to preventingor reducing the direct or indirect association of one or more molecules,peptides, proteins, enzymes, or receptors, or preventing or reducing thenormal activity of one or more molecules, peptides, proteins, enzymes orreceptors, e.g., preventing or reducing the direct or indirectassociation with human ERR-α.

As used herein, the term “competitive inhibitor” refers to inhibitorsthat bind to human ERR-α at active site, thus directly competing with asubstrate or ligand. Competitive inhibition may, in some instances, bereversed completely by increasing the substrate or ligand concentration.

As used herein, the term “uncompetitive inhibitor” refers to one thatinhibits the functional activity of human ERR-α by binding to adifferent site than does its substrate(s).

As used herein, the term “non-competitive inhibitor” refers to one thatcan bind to either the free or bound form of ERR-α. Those of skill inthe art may identify inhibitors as competitive, uncompetitive, ornon-competitive by computer fitting enzyme kinetic data using standardmethods. See, for example, (Segel 1975).

The term “inverse agonist” as used herein refers to compounds orsubstances that have the ability to decrease the constitutive level ofreceptor activation in the absence of an agonist instead of onlyblocking the activation induced by agonist binding at the receptor.

As used herein, the term “R or S-isomer” refers to two possiblestereoisomers of a chiral carbon according to the Cahn-Ingold-Prelogsystem adopted by International Union of Pure and Applied Chemistry(IUPAC). Each group attached to the chiral carbon is first assigned to apreference or priority a, b, c, or d on the basis of the atomic numberof the atom that is directly attached to the chiral carbon. The groupwith the highest atomic number is given the highest preference a, thegroup with next highest atomic number is given the next highestpreference b, and so on. The group with the lowest preference (d) isthen directed away from the viewer. If the trace of a path from a to bto c is counter clockwise, the isomer is designated (S); in the oppositedirection, clockwise, the isomer is designated (R).

As used herein, the term “stereoisomers” is a general term for allisomers of individual molecules that differ only in the orientation oftheir atoms in space. It includes enantiomers and isomers of compoundswith more than one chiral center that are not mirror images of oneanother (diastereomers).

As used herein, the term “chiral center” refers to a carbon atom towhich four different groups are attached.

As used herein, the term “enantiomer” or “enantiomeric” refers to amolecule that is nonsuperimposable on its mirror image and henceoptically active wherein the enantiomer rotates the plane of polarizedlight in one direction and its mirror image rotates the plane ofpolarized light in the opposite direction.

As used herein, the term “racemic” refers to a mixture of equal parts ofenantiomers and which is optically active.

As used herein, the term “resolution” refers to the separation orconcentration or depletion of one of the two enantiomeric forms of amolecule. In the context of this application. The term “resolution” alsorefers to the amount of detail, which can be resolved by the diffractionexperiment. Or in other terms, since the inherent disorder of a proteincrystal diffraction pattern fades away at some diffraction angletheta_(max), the corresponding distance d_(min) of the reciprocallattices is determined by Bragg's law. In practice in proteincrystallography it is usual to quote the nominal resolution of a proteinelectron density in terms of d_(min), the minimum lattice distance towhich data is included in the calculation of the map.

As used herein, the term “ligand” refers to any molecule, or chemicalentity, which binds with or to ERR-α, a subunit of ERR-α, a domain ofERR-α, a target structural motif of ERR-α, or a fragment of ERR-α. Thus,ligands include, but are not limited to, modulators of ERR-α activitysuch as small molecule inhibitors, small molecule agonists, and smallmolecule inverse agonists, for example.

As used herein, the term “small molecule inhibitor” refers to ligandsuseful in the present invention having the ability to modulate ameasurable amount of ERR-α activity. In addition to small organicmolecules, peptides, antibodies, cyclic peptides and peptidomimetics arecontemplated as being useful in the disclosed methods. Preferredinhibitors and modulators are small molecules, preferably less than10,000 daltons, and more preferably less than 5,000 daltons.

As used herein the terms “bind”, “binding”, “bond”, or “bonded” whenused in reference to the association of atoms, molecules, or chemicalgroups, refer to any physical contact or association of two or moreatoms, molecules; or chemical groups.

As used herein, the terms “covalent bond” or “valence bond” refer to achemical bond between two atoms in a molecule created by the sharing ofelectrons, usually in pairs, by the bonded atoms.

As used herein, “noncovalent bond” refers to an interaction betweenatoms and/or molecules that does not involve the formation of a covalentbond between them.

The term “composition” is intended to encompass a product comprising thespecified ingredients in the specified amounts, as well as any productwhich results, directly or indirectly, from combinations of thespecified ingredients in the specified amounts.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who is the object of treatment,observation or experiment.

It is intended that the definition of any substituent or variable at aparticular location in a molecule be independent of its definitionselsewhere in that molecule. It is understood that substituents andsubstitution patterns on the compounds of this invention can be selectedby one of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art as well as those methods set forth herein.

Metabolic disorders, diseases, or conditions include, but are notlimited to, diabetes, obesity, and associated symptoms or complicationsthereof. They include such conditions as IDDM (insulin-dependentdiabetes mellitus), NIDDM (non insulin-dependent diabetes mellitus), IGT(Impaired Glucose Tolerance), IFG (Impaired Fasting Glucose), Syndrome X(or Metabolic Syndrome), hyperglycemia, elevated blood glucose level,and insulin resistance. A condition such as IGT or IFG is also known asa “prediabetic condition” or “prediabetic state”.

Methods are known in the art for determining effective doses fortherapeutic and prophylactic purposes for the disclosed pharmaceuticalcompositions or the disclosed drug combinations, whether or notformulated in the same composition. For therapeutic purposes, the term“therapeutically effective amount” as used herein, means that amount ofeach active compound or pharmaceutical agent, alone or in combination,that elicits the biological or medicinal response in a tissue system,animal or human that is being sought by a researcher, veterinarian,medical doctor or other clinician, which includes alleviation of thesymptoms of the disease or disorder being treated. For prophylacticpurposes (i.e., inhibiting the onset or progression of a disorder), theterm “therapeutically effective amount” refers to that amount of eachactive compound or pharmaceutical agent, alone or in combination, thattreats or inhibits in a subject the onset or progression of a disorderas being sought by a researcher, veterinarian, medical doctor or otherclinician. Thus, the present invention provides combinations of two ormore drugs wherein, for example, (a) each drug is administered in anindependently therapeutically or prophylactically effective amount; (b)at least one drug in the combination is administered in an amount thatis sub-therapeutic or sub-prophylactic if administered alone, but istherapeutic or prophylactic when administered in combination with thesecond or additional drugs according to the invention; or (c) both (ormore) drugs are administered in an amount that is sub-therapeutic orsub-prophylactic if administered alone, but are therapeutic orprophylactic when administered together.

The term “pharmaceutically acceptable salt” refers to non-toxicpharmaceutically acceptable salts (Berge, Bighley et al. 1977; Gould1986). Other salts well known to those in the art may, however, beuseful in the preparation of compounds according to this invention or oftheir pharmaceutically acceptable salts. Representative organic orinorganic acids include, but are not limited to, hydrochloric,hydrobromic, hydriodic, perchloric, sulfuric, nitric, phosphoric,acetic, propionic, glycolic, lactic, succinic, maleic, fumaric, malic,tartaric, citric, benzoic, mandelic, methanesulfonic,hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic,2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic,salicylic, saccharinic or trifluoroacetic acid. Representative organicor inorganic bases include, but are not limited to, basic or cationicsalts such as benzathine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine, procaine, aluminum, calcium, lithium,magnesium, potassium, sodium and zinc.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

It is to be understood at the outset, that the figures and examplesprovided herein are to exemplify, and not to limit the invention and itsvarious embodiments.

The present invention includes a crystal comprising a complex ofEstrogen Related Receptor alpha (ERR-α), or a fragment, or targetstructural motif or derivative thereof, and a ligand, wherein the ligandthat forms a thioether bond to Cys325 of ERR-α. In a preferredembodiment, the fragment or derivative thereof is a peptide comprisingSEQ ID NO:2. In another preferred embodiment, the ligand is Compound 1,or derivatives thereof. In highly preferred embodiment, the crystal hasa spacegroup of P6522. In another highly preferred embodiment, thecrystal comprises a unit cell consisting of about a=b=103.007 andc=110.017. In yet another highly preferred embodiment, the crystalcomprises a complex of SEQ ID NO:2 and Compound 1 comprising an atomicstructure characterized by the coordinates of Table 6.

The present invention also includes a crystal comprising a complex ofERR-α and a ligand that forms a thioether bond to Cys325 of ERR-α, inwhich ERR-α comprises a peptide having at least 95% sequence identity toSEQ ID NO:2.

In another aspect of the invention, the invention includes a computersystem comprising: (a) a database stored on a computer readable storagemedium containing information on the three dimensional structure of acrystal comprising a complex of ERR-α, or a fragment or a targetstructural motif or derivative thereof, and a ligand, wherein the ligandthat forms a thioether bond to Cys325 of ERR-α; and, (b) a userinterface to view the information. In one embodiment, the informationcomprises diffraction data obtained from a crystal comprising a complexof SEQ ID NO:2 and a ligand that forms a thioether bond to Cys325 ofERR-α. In a preferred embodiment, the information comprises diffractiondata obtained from a crystal comprising a complex of SEQ ID NO:2 andCompound 1, or derivatives thereof. In a highly preferred embodiment,the information comprises diffraction data characterized by thecoordinates of Table 6.

In another embodiment, the information comprises an electron density mapof a crystal form comprising a complex of SEQ ID NO:2 and a ligand thatforms a thioether bond to SEQ ID NO:2. In a preferred embodiment, theinformation comprises an electron density map of a crystal comprising acomplex of SEQ ID NO:2 and Compound 1, or derivatives thereof. In ahighly preferred embodiment, the information comprises an electrondensity map derived from the structure coordinates of Table 6, orhomologous structure coordinates comprising a root mean square deviationof non-hydrogen atoms of less than about 1.5 Å when superimposed on thenon-hydrogen atom positions of the corresponding atomic coordinates ofTable 6. In a preferred embodiment, the information comprises structurecoordinates comprising a root mean square deviation of non-hydrogenatoms of less than about 0.75 Å when superimposed on the non-hydrogenatom positions of the corresponding atomic coordinates of Table 6.

The present invention also includes a method of evaluating the potentialof a ligand to modulate the activity of ERR-α comprising the steps of:(a) exposing ERR-α to the ligand; and (b) detecting the formation of athioether bond to Cys325 of ERR-α, thereby evaluating the potential ofthe ligand to modulate the activity of ERR-α. In one embodiment of themethod of the invention described above, the ligand is a virtualcompound. In another embodiment the present invention includes a methodof evaluating the potential of a ligand to modulate the activity ofERR-α comprising the steps of: (a) comparing the atomic structure of theligand to the three dimensional structure of a complex of ERR-α and aligand that forms a thioether bond to Cys325 of ERR-α; and, (b) usingthe information obtained in step (a) to determine if the ligand couldform a thioether bond to Cys325 of ERR-α, thereby evaluating thepotential of the ligand to modulate the activity of ERR-α. In adifferent embodiment the present invention includes a method ofevaluating the potential of a ligand to modulate the activity of ERR-αcomprising the steps of: (a) comparing the atomic structure of theligand to the three dimensional structure of a complex of ERR-α and aligand that forms a thioether bond to Cys325 of ERR-α, wherein thecomparing comprises employing a computational means to perform a fittingoperation between the ligand and a binding site of ERR-α, wherein thebinding site is defined by structure coordinates for Cys325 according toTable 6; and, (b) using the information obtained in step (a) todetermine if the ligand could form a thioether bond to Cys325 of ERR-α,thereby evaluating the potential of the ligand to modulate the activityof ERR-α. In a highly preferred embodiment, the present inventionincludes a method of evaluating the potential of a ligand to modulatethe activity of ERR-α comprising the steps of: (a) exposing the ligandto crystalline SEQ ID NO:2; (b) detecting the formation of a thioetherbond to Cys325 of ERR-α by determining the three dimensional structureof the complex of SEQ ID NO:2 and the ligand that formed a thioetherbond to SEQ ID NO:2; thereby evaluating the potential of the ligand tomodulate the activity of ERR-α. In a preferred embodiment, the ligandmodulates the activity of ERR-α by functioning as an inverse agonist ofERR-α.

The present invention includes a method of identifying a ligand with theability to modulate the activity of ERR-α, comprising the step of; (a)using the three dimensional structure of ERR-α cocrystallized with asmall molecule that forms a thioether bond to Cys325 of ERR-α to designor select said ligand; (b) designing or selecting said ligand; therebyidentifying a ligand with the ability to modulate the activity of ERR-α.In one embodiment, the three dimensional structure corresponds to theatomic structure characterized by the coordinates of Table 6, or similarstructure coordinates comprising a root mean square deviation ofnon-hydrogen atoms of less than about 1.5 Å when superimposed on thenon-hydrogen atom positions of the corresponding atomic coordinates ofTable 6. In a different embodiment, the method described above furthercomprises the steps of: (c) synthesizing the ligand; and (d) contactingthe ligand with ERR-α. In a preferred embodiment, the ligand is aninverse agonist of ERR-α.

The instant invention comprises a method of locating the attachment siteof a small molecule ligand that modulates the activity of ERR-α,comprising the steps of: (a) obtaining X-ray diffraction data for acrystal of ERR-α; (b) obtaining X-ray diffraction data for a complex ofERR-α and small molecule ligand that forms a thioether bond to Cys325 ofERR-α; (c) subtracting the X-ray diffraction data obtained in step (a)from the X-ray diffraction data obtained in step (b) to obtain thedifference in the X-ray diffraction data; (d) obtaining phases thatcorrespond to X-ray diffraction data obtained in step (a); (e) utilizingthe phases obtained in step (d) and the difference in the X-raydiffraction data obtained in step (c) to compute a difference Fourierimage of the small molecule ligand; and, (f) locating the attachmentsite of the small molecule ligand to ERR-α based on the computationsobtained in step (e). In a preferred embodiment, the small moleculeligand is an inverse agonist of ERR-α and the attachment site is Cys325of ERR-α.

In another aspect of the present invention, the invention includes amethod for the production of a crystal complex comprising an ERR-αpolypeptide and a ligand that forms a thioether bond to Cys325 of ERR-α,comprising the steps of: (a) contacting the ERR-α polypeptide with saidligand in a suitable solution comprising ammonium sulfate, Pipes pH 6.5and Na-thiocyanate; and, (b) crystallizing said resulting complex ofERR-α polypeptide and the ligand from said solution. In one embodiment,the ERR-α polypeptide is a polypeptide SEQ ID NO:2. In a preferredembodiment, the ligand is Compound 1, or a derivative thereof.

The invention further includes a method for the production of a crystalcomprising a complex of ERR-α and a ligand that forms a thioether bondto Cys325 of ERR-α, wherein the ligand is a small molecule ligand thatfunctions as an inverse agonist of ERR-α, comprising the steps of: (a)contacting and ERR-α polypeptide with said ligand; and, (b)cocrystallizing the polypeptide comprising SEQ ID NO:2 with the ligand;thereby producing the crystal comprising a complex of ERR-α and theligand that forms the thioether bond to Cys325 of ERR-α.

The invention includes a method for identifying a ligand that functionsas an inverse agonist of ERR-α comprising the steps of: (a) using athree dimensional structure of the complex of ERR-α and Compound 1, asdefined by atomic coordinates according to Table 6; (b) replacing one ormore ERR-α amino acids in said three-dimensional structure with adifferent amino acid to produce a modified ERR-α; (c) using saidthree-dimensional structure to design or select said ligand; (d)synthesizing said ligand; and, (e) contacting said ligand with saidmodified ERR-α in an assay designed to test the ability of the ligand tomodulate the activity of ERR-α or said modified ERR-α. In anotherembodiment, the ligand identified in the method described above isselected from a database. In a preferred embodiment, the ligandidentified in the method described above is designed de novo. In anotherpreferred embodiment, the ligand identified in the method describedabove is designed from a known inverse agonist or other type ofmodulator.

Engineered Forms and Fragments

Engineered forms of ERR-α or fragments thereof, for instance engineeredforms or fragments comprising active sites defined by two or more aminoacids may be prepared by any available means including synthetic orrecombinant means. Such fragments may then be used in the assays asdescribed herein, for example, but not limited to, high-throughputassays to detect interactions between prospective ligands and the activesite within the fragment.

For recombinant expression or production of the forms or fragments ofthe invention, nucleic acid molecules encoding the form or fragment maybe prepared. Nucleic acid molecules encoding engineered forms orfragments of the invention may differ in sequence because of thedegeneracy in the genetic code or may differ in sequence as they encodeproteins or protein fragments that differ in amino acid sequence.Homology or sequence identity between two or more such nucleic acidmolecules is determined by BLAST (Basic Local Alignment Search Tool)analysis using the algorithm employed by the programs blastp, blastn,blastx, tblastn and tblastx (Karlin and Altschul 1990) and (Altschul1993), fully incorporated by reference, which are tailored for sequencesimilarity searching.

The approach used by the BLAST program is to first consider similarsegments between a query sequence and a database sequence, then toevaluate the statistical significance of all matches that are identifiedand finally to summarize only those matches which satisfy a preselectedthreshold of significance. For a discussion of basic issues insimilarity searching of sequence databases, see (Altschul, Boguski etal. 1994) which is fully incorporated by reference. The searchparameters for histogram, descriptions, alignments, expect (i.e., thestatistical significance threshold for reporting matches againstdatabase sequences), cutoff, matrix and filter are at the defaultsettings. For a discussion of default scoring matrix used by blastp,blastx, tblastn, and tblastx, see (Henikoff 1992).

The encoding nucleic acid molecules of the present invention orfragments thereof (i.e., synthetic oligonucleotides) and those that areused as probes or specific primers for polymerase chain reaction (PCR)or to synthesize gene sequences encoding proteins of the invention caneasily be synthesized by chemical techniques, for example, thephosphotriester method of (Matteucci and Caruthers 1981) or usingautomated synthesis methods. In addition, larger DNA segments canreadily be prepared by well-known methods, such as synthesis of a groupof oligonucleotides that define various modular segments of the gene,followed by ligation of oligonucleotides to build the complete modifiedgene.

The encoding nucleic acid molecules of the present invention may furtherbe modified so as to contain a detectable label for diagnostic and probepurposes. A variety of such labels are known in the art and can readilybe employed with the encoding molecules herein described. Suitablelabels include, but are not limited to, biotin, radiolabeled nucleotidesand the like. A skilled artisan can employ any of the art-known labelsto obtain a labeled encoding nucleic acid molecule.

The present invention further provides recombinant DNA molecules (rDNA)that contain a coding sequence for a protein or protein fragment asdescribed herein. As used herein, an rDNA molecule is a DNA moleculethat has been subjected to molecular manipulation. Methods forgenerating rDNA molecules are well known in the art, for example, see(Sambrook, Fritsch et al. 1989). In the preferred rDNA molecules, acoding DNA sequence is operably linked to expression control sequencesand/or vector sequences.

The choice of vector and expression control sequences to which one ofthe protein encoding sequences of the present invention is operablylinked depends directly, as is well known in the art, on the functionalproperties desired (e.g., protein expression, and the host cell to betransformed). A vector of the present invention may be capable ofdirecting the replication or insertion into the host chromosome, andpreferably also expression, of the structural gene included in the rDNAmolecule.

Expression control elements that are used for regulating the expressionof an operably linked protein encoding sequence are known in the art andinclude, but are not limited to, inducible promoters, constitutivepromoters, secretion signals, and other regulatory elements. Preferably,the inducible promoter is readily controlled, such as being responsiveto a nutrient in the host cell's medium.

The present invention further provides host cells transformed with anucleic acid molecule that encodes a protein or protein fragment of thepresent invention. The host cell can be either prokaryotic oreukaryotic. Eukaryotic cells useful for expression of a protein of theinvention are not limited, so long as the cell line is compatible withcell culture methods and compatible with the propagation of theexpression vector and expression of the gene product. Preferredeukaryotic host cells include, but are not limited to, insect, yeast,and mammalian cells. Preferred eukaryotic host cells include Spodopterafrugiperda (Sf9 or Sf21) insect cells.

Transformed host cells of the invention may be cultured under conditionsthat allow the production of the recombinant protein. Optionally therecombinant protein is isolated from the medium or from the cells;recovery and purification of the protein may not be necessary in someinstances where some impurities may be tolerated.

Kits may also be prepared with any of the above described nucleic acidmolecules, proteins, protein fragments, vector and/or host cellsoptionally packaged with the reagents needed for a specific assay, suchas those described above. In such kits, the protein, protein fragments,or other reagents may be attached to a solid support, such as glass orplastic beads.

High-Throughput Assays

Compound identification methods can be performed using conventionallaboratory assay formats or in high-throughput assays, including, butnot limited to, those described below.

Immunoassays are a group of techniques used for the measurement ofspecific biochemical substances, commonly at low concentrations incomplex mixtures such as biological fluids. The assays depend uponsuitably prepared and selected antibodies with specificity and highaffinity for their complementary antigens. A substance to be measuredmust, of necessity, be antigenic, either an immunogenic macromolecule ora haptenic small molecule. To each sample a known limited amount ofspecific antibody is added and the fraction of the antigen combiningwith it; often expressed as the bound:free ratio, is estimated byquantifying the signal from the antibody. Quantification can be achievedwith a number of readily identifiable labels and used for various typesof assays, including, but not limited to, radioisotopes forradioimmunoassays (RIA), fluorescent molecules for fluoroimmunoassays(FIA), stable free radicals for spin immunoassays, chemiluminescentmolecules for chemiluminescent immunoassays (CLIA), colloidal goldparticles for immunogold assays, and enzymes for enzyme-linkedimmunosorbent assays (ELISA).

A common immunoassay format is the ELISA, which avoids the hazards ofradiochemicals and the expense of fluorescence detection systems.Instead, an ELISA is a form of quantitative immunoassay based on the useof antibodies (or antigens) that may be linked to an insoluble carriersurface, which is then used to “capture” the relevant antigen (orantibody) the test solution. The antigen-antibody complex is thendetected by measuring the activity of an appropriate enzyme that can becovalently attached to the capture antigen (or antibody) or to asubsequent “detection” antibody (or antigen). For more information onELISA techniques, see, for example, (Crowther 1995); (Kemeny (editor)and Challacombe (editor) 1988), (Kemeny 1991), and (Ishikawa 1999).

Colorimetric assays for enzymes are methods of quantitative chemicalanalysis in which the concentration or amount of a compound isdetermined by comparing the color produced by the reaction of a reagentwith both standard and test amounts of the compound, often using acolorimeter. A colorimeter is a device for measuring color intensity ordifferences in color intensity, either visually or photoelectrically.Standard colorimetric assays of beta-galactosidase enzymatic activityare well known to those skilled in the art, see for example, (Norton andCoffin 1985). A colorimetric assay can be performed on whole celllysates using O-nitrophenyl-beta-D-galacto-pyranoside (ONPG, Sigma) asthe substrate in a standard colorimetric beta-galactosidase assay(Sambrook, Fritsch et al. 1989). Automated colorimetric assays are alsoavailable for the detection of beta-galactosidase activity, as describedin U.S. patent Number (U.S. Pat. No. 5,733,720).

Enzymatic substrates that become fluorescent after being acted upon byan enzyme generally are well known. Such fluorescent substratestypically have two components that are bound to one another through, forexample, a covalent chemical bond. One component is a fluorescentmolecule that is capable of fluorescing by first accepting light energyand then emitting light energy. The other component is an entity thatprevents the fluorescent molecule from accepting or emitting lightenergy when the two components are covalently bound to one another. Inthe presence of an appropriate enzyme, the enzyme cleaves the covalentbond between the two components and separates one component from theother to permit the fluorescent molecule to accept and emit lightenergy. In other words, the enzyme frees the fluorescent molecule andallows it to fluoresce. Ideally, fluorescent substrates should besoluble and stable in aqueous buffers, should have a high affinity forthe enzymes that act upon them, and should yield a strong signal uponenzymatic action (U.S. Pat. No. 5,998,593A).

Detecting fluorescence emitted from the fluorescent component of afluorescent enzyme substrate is typically achieved in two steps. Thefluorescent molecule is first excited with light energy and subsequentlythe fluorescence emitted from the fluorescent component is thendetected. Generally, fluorescent molecules can be excited with lightenergy from, for example, a laser or another suitable light source.Fluorescence is detected with a device designed to detect light energyof a wavelength that is emitted by the fluorescent molecule. Suchexcitation and emission detection systems generally are designed tooperate at particular wavelength ranges (U.S. Pat. No. 5,998,593A).

Time-resolved Fluorescence resonance energy transfer (TR-FRET) unitesTRF (Time-Resolved Fluorescence) and FRET (Fluorescence Resonance EnergyTransfer) principles. This combination brings together the lowbackground benefits of TRF with the homogeneous assay format of FRET.Time-resolved fluorometry (TRF) takes advantage of the unique propertiesof the rare earth elements called lanthanides. Specifically, lanthanideshave large Stoke's shifts and extremely long emission half-livescompared to more traditional fluorophores. The commonly used lanthanidesin TRF assays are samarium (Sm), europium (Eu), terbium (Tb), anddysprosium (Dy). Lanthanides are complexed with organic moieties thatharvest light and transfer it to the lanthanide through intramolecularprocesses. FRET uses two fluorophores, a donor and an acceptor.Excitation of the donor by an energy source (e.g. flash lamp orfluorometer laser) triggers an energy transfer to the acceptor if theyare within a given proximity to each other. The acceptor in turn emitslight at its given wavelength. Because of this energy transfer,molecular interactions between biomolecules can be assessed by couplingeach partner with a fluorescent label and detecting the level of energytransfer. More importantly acceptor emissions, as a measure of energytransfer, can be detected without the need to separate bound fromunbound assay components (Klostermeier and Millar 2001).

Thermofluor® assays detect small changes in the intrinsic meltingtemperature of proteins based on binding of ligands. Compounds thatinteract preferentially with the native form of the protein willincrease the T_(m), the temperature at which half of the protein isunfolded (Pantoliano, Petrella et al. 2001). The technique monitorschanges in the fluorescent intensity of dyes such as1-anilinonaphthalene-8-sulfonic acid (1,8-ANS). The fluorescent dyes arequenched in aqueous environments but increase in fluorescence on bindingto the hydrophobic core of denatured proteins.

Modeling the Three-Dimensional Structure of ERR-α

The atomic coordinate data provided in Table 6, or the coordinate dataderived from homologous proteins may be used to build athree-dimensional model of ERR-α. Any available computational methodsmay be used to build the three dimensional model. As a starting point,the X-ray diffraction pattern obtained from the assemblage of themolecules or atoms in a crystalline version of ERR-α or an ERR-α homologcan be used to build an electron density map using tools well known tothose skilled in the art of crystallography and X-ray diffractiontechniques. Additional phase information extracted either from thediffraction data and available in the published literature and/or fromsupplementing experiments may then be used to complete thereconstruction.

For basic concepts and procedures of collecting, analyzing, andutilizing X-ray diffraction data for the construction of electrondensities see, for example, (Campbell 1984), (Cantor and Schimmel 1980),(Brunger 1993), (Woolfson 1997), (Drenth 1999), (Tsirelson and Ozerov1996), and U.S. patent Numbers (U.S. Pat. No. 5,942,428A); (U.S. Pat.No. 6,037,117A); (U.S. Pat. No. 5,200,910A); and (U.S. Pat. No.5,365,456A), each of which is herein specifically incorporated byreference in their entirety.

For basic information on molecular modeling, see, for example, (Schlecht1998); (Gans, Amann et al. 1996); (Cohen (editor) 1996); and (Smith1996). U.S. patents which provide detailed information on molecularmodeling include U.S. patent Numbers (U.S. Pat. No. 4,906,122A; U.S.Pat. No. 5,030,103A; U.S. Pat. No. 5,583,973A; U.S. Pat. No. 5,612,894A;U.S. Pat. No. 5,994,503A; U.S. Pat. No. 6,071,700A; U.S. Pat. No.6,075,014A; U.S. Pat. No. 6,075,123A; U.S. Pat. No. 6,080,576A; U.S.Pat. No. 6,093,573A), each of which are incorporated by reference hereinin their entirety.

Methods of Using the Atomic Coordinates to Identify and Design Ligandsof Interest

The atomic coordinates of the invention, such as those described inTable 6, or coordinates substantially identical to or homologous tothose of Table 6 may be used with any available methods to prepare threedimensional models of ERR-α as well as to identify and design ERR-αligands, inhibitors, antagonists, agonist, or inverse agonist molecules.Such a method provides the amino acid sequence and/or X-ray diffractiondata in a form which allows a skilled artisan to analyze and molecularmodel the three-dimensional structure of ERR-α or related molecules,including a subdomain thereof.

For instance, three-dimensional modeling may be performed using theexperimentally determined coordinates derived from X-ray diffractionpatterns, such as those in Table 6, for example, wherein such modelingincludes, but is not limited to, drawing pictures of the actualstructures, building physical models of the actual structures, anddetermining the structures of related subunits and ERR-α:ligand andERR-α subunit:ligand complexes using the coordinates. Such molecularmodeling can utilize known X-ray diffraction molecular modelingalgorithms or molecular modeling software to generate atomic coordinatescorresponding to the three-dimensional structure of ERR-α.

As described above, molecular modeling involves the use of computationalmethods, preferably computer assisted methods, to build realistic modelsof molecules that are identifiably related in sequence to the knowncrystal structure. It also involves modeling new small molecules boundto ERR-α starting with the structures of ERR-α and or ERR-α complexedwith known ligands or other molecules. The methods utilized in ligandmodeling range from molecular graphics (i.e., 3D representations) tocomputational chemistry (i.e., calculations of the physical and chemicalproperties) to make predictions about the binding of ligands oractivities of ligands; to design new ligands; and to predict novelmolecules, including ligands such as drugs, for chemical synthesis,collectively referred to as rational drug design.

One approach to rational drug design is to search for known molecularstructures that might bind to an active site. Using molecular modeling,rational drug design programs can look at a range of different molecularstructures of drugs that may fit into the active site of an enzyme, andby moving them in a three-dimensional environment it can be decidedwhich structures actually fit the site well.

An alternative but related rational drug design approach starts with theknown structure of a complex with a small molecule ligand and modelsmodifications of that small molecule in an effort to make additionalfavourable interactions with ERR-α.

The present invention includes the use of molecular and computermodeling techniques to design and select and design ligands, such assmall molecule ligands that act as agonists, antagonists, inverseagonists or other therapeutic agents that interact with ERR-α. Forexample, the invention as herein described includes the design ofligands that act as modulators of at least one ERR-α function by bindingto all, or a portion of, the active sites or other regions of ERR-α. Ina preferred embodiment, the ligand binds to Cys325 of ERR-α. In anotherpreferred embodiment the ligand is an inverse agonist. Similarly, agentsthat modulate at least one function of ERR-α, whether or not it is boundto another chemical entity, may be designed using the atomic coordinatesof ERR-α or complexes comprising ERR-α of this invention.

The atomic coordinates of the present invention also provide the neededinformation to probe a crystal of ERR-α with molecules composed of avariety of different chemical features to determine optimal sites forinteraction between candidate modulators of ERR-α activity and ERR-α.For example, high resolution X-ray diffraction data collected fromcrystals saturated with solvent allows the determination of where eachtype of solvent molecule sticks. Small molecules that bind to thosesites can then be designed and synthesized and tested for their abilityto modulate activity (Travis 1993).

The present invention also includes methods for computationallyscreening small molecule databases and libraries for chemical entities,agents, ligands, or compounds that can bind in whole, or in part, toERR-α. In this screening, the quality of fit of such entities orcompounds to the binding site or sites may be judged either by shapecomplementarity or by estimated interaction energy (Meng, Shoichet etal. 1992).

The design of ligands that bind to, promote or inhibit the functionalactivity of ERR-α according to this invention generally involvesconsideration of two factors. First, the compound must be capable ofphysically and structurally associating with ERR-α. In addition to thecovalent interaction described herein, non-covalent molecularinteractions important in the association of ERR-α with the ligandinclude hydrogen bonding, van der Waals and hydrophobic interactions.Second, the ligand must be able to assume a conformation that allows itto associate with ERR-α. Although certain portions of the ligand may notdirectly participate in the association with ERR-α, those portions maystill influence the overall conformation of the molecule. This, in turn,may have a significant impact on binding affinities, therapeuticefficacy, drug-like qualities and potency of the ligand. Suchconformational requirements include the overall three-dimensionalstructure and orientation of the ligand in relation to all or a portionof the active site or other region of ERR-α, or the spacing betweenfunctional groups of a ligand comprising several chemical entities thatdirectly interact with ERR-α.

The potential, predicted, agonist, antagonist, inverse agonist, orbinding effect of a ligand or other compound on ERR-α may be analyzedprior to its actual synthesis and testing by the use of computermodeling techniques. If the theoretical structure of the given ligandsuggests insufficient interaction and association between it and ERR-α,synthesis and testing of the ligand may be obviated. If computermodeling indicates a strong interaction, however, the molecule may thenbe synthesized and tested for its ability to interact with ERR-α. Inthis manner, synthesis of inoperative ligand may be avoided. In somecases, inactive ligands are synthesized predicted on modeling and thentested to develop a SAR (structure-activity relationship) for compoundsinteracting with a specific region of ERR-α.

One skilled in the art may use one of several methods to screen chemicalentities, fragments, compounds, or other agents for use as ligands basedon their ability to associate with ERR-α and more particularly theirability to associate with the individual binding pockets or active sitesof ERR-α. This process may begin by visual inspection of, for example,the active site on the computer screen based on the atomic coordinatesof ERR-α or ERR-α complexed with a ligand. Selected chemical entities,compounds, or agents may then be positioned in a variety oforientations, or docked within an individual binding pocket of ERR-α.Docking may be accomplished using software such as QUANTA, availablefrom Accelrys, Inc., San Diego, Calif.; and SYBYL, available for Tripos,St. Louis, Mo.; followed by energy minimization and molecular dynamicswith standard molecular mechanics forcefields, such as CHARMm; availablefrom Accelrys, Inc., San Diego, Calif.; and AMBER, University ofCalifornia, San Francisco.

Specialized computer programs may also assist in the process ofselecting chemical entities. These include but are not limited to: GRID(Goodford 1985), available from Oxford University, Oxford, UK); MCSS(Miranker and Karplus 1991), available from Molecular Simulations,Burlington, Mass.; AUTODOCK (Goodsell and Olsen 1990), available fromScripps Research Institute, La Jolla, Calif.; and DOCK (Kuntz, Blaney etal. 1982), available from University of California, San Francisco,Calif.

The use of software such as GRID, a program that determines probableinteraction sites between probes with various functional groupcharacteristics and the macromolecular surface, is used to analyze thesurface sites to determine structures of similar inhibiting proteins orcompounds. The GRID calculations, with suitable inhibiting groups onmolecules (e.g., protonated primary amines) as the probe, are used toidentify potential hotspots around accessible positions at suitableenergy contour levels. The program DOCK may be used to analyze an activesite or ligand-binding site and suggest ligands with complementarysteric properties.

Once suitable chemical entities, compounds, or agents have been selectedas potential ligands, they can be assembled into a single ligand,compound, antagonist (inhibitor), agonist (activator), or inverseagonist. Assembly may proceed by visual inspection of the relationshipof the fragments to each other on the three-dimensional image. This maybe followed by manual model building using software such as QUANTA orSYBYL.

Useful programs to aid in connecting the individual chemical entities,compounds, or agents include but are not limited to: CAVEAT (Bartlett,Shea et al. 1989); 3D Database systems such as MACCS-3D (Martin 1992),available from MDL Information Systems, San Leandro, Calif.; and HOOK,available from Molecular Simulations, Burlington, Mass.

Several methodologies for searching three-dimensional databases to testpharmacophore hypotheses and select compounds for screening areavailable. These include the program CAVEAT (Bacon and Moult 1992). Forinstance, CAVEAT uses databases of cyclic compounds which can act as“spacers” to connect any number of chemical fragments already positionedin the active site. This allows one skilled in the art to quicklygenerate hundreds of possible ways to connect the fragments alreadyknown or suspected to be necessary for tight binding.

Instead of proceeding to build an inhibitor, activator, agonist,antagonist, or inverse agonist of ERR-α in a step-wise fashion, onechemical entity at a time as described above, such ligands may bedesigned as a whole or “de novo” using either an empty active site oroptionally including some portion(s) of a known molecule(s). Thesemethods include: LUDI (Bohm 1992), available from Biosym Technologies,San Diego, Calif.; LEGEND (Nishibata and Itai 1991), available fromMolecular Simulations, Burlington, Mass.; and LeapFrog, available fromTripos Associates, St. Louis, Mo., USA.

For example, the program LUDI can determine a list of interaction sitesinto which to place both hydrogen bonding and hydrophobic fragments.LUDI then uses a library of linkers to connect up to four differentinteraction sites into fragments. Then smaller “bridging” groups such as—CH₂— and —COO— are used to connect these fragments. For the enzymeDHFR, the placements of key functional groups in the well-knowninhibitor methotrexate were reproduced by LUDI. See also, (Rotstein andMurcko 1993).

Other molecular modeling techniques may also be employed in accordancewith this invention. See, e.g., (Cohen, Blaney et al. 1990). See also,(Navia and Murcko 1992).

Once a ligand has been designed or selected by the above methods, theaffinity with which that ligand may bind or associate with ERR-α may betested and optimized by computational evaluation and/or by testingbiological activity after synthesizing the compound. Ligands mayinteract with the ERR-α in more than one conformation that is similar inoverall binding energy. In those cases, the deformation energy ofbinding is taken to be the difference between the energy of the freeligand and the average energy of the conformations observed when theligand binds to ERR-α.

A ligand designed or selected as binding or associating with ERR-α maybe further computationally optimized so that in its bound state it wouldpreferably lack repulsive electrostatic interaction with ERR-α. Suchnon-complementary (e.g., electrostatic) interactions include repulsivecharge-charge, dipole-dipole and charge-dipole interactions.Specifically, the sum of all electrostatic interactions between thecompound and ERR-α when the compound is bound, preferably make a neutralor favourable contribution to the enthalpy of binding. Weak bindingcompounds will also be designed by these methods so as to determine SAR.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interaction. Examples of programsdesigned for such uses include: Gaussian 92, revision C (Frisch, Truckset al. 1992); AMBER, University of California, San Francisco; QUANTA andCHARMm, available from Accelrys, Inc., San Diego, Calif.; and InsightII/Discover, from Biosysm Technologies Inc., San Diego, Calif., USA.Other hardware systems and software packages will be known to thoseskilled in the art.

Once a ligand that associates with ERR-α has been optimally selected ordesigned, as described above, substitutions may then be made in some ofits atoms or side groups in order to improve or modify its bindingproperties. Generally, initial substitutions are conservative, i.e., thereplacement group will have approximately the same size, shape,hydrophobicity and charge as the original group. It should, of course,be understood that components known in the art to alter conformation maybe avoided. Such substituted ligands may then be analyzed for efficiencyof fit to ERR-α by the same computer methods described in detail, above.

Use of Homology Structure Modeling to Design Ligands with ModulatedBinding or Activity to ERR-α.

The present invention includes the use of the atomic coordinates andstructures of ERR-α complexed with a ligand that forms a thioether bondto Cys325 of ERR-α to design modifications to starting ligands andderivatives thereof that will bind more tightly or interact morespecifically to the target enzyme.

The structure of a complex between the ERR-α and the starting ligand canbe used to guide the modification of that ligand to produce new ligandsthat have other desirable properties for applicable industrial and otheruses (e.g., as pharmaceuticals), such as chemical stability, solubilityor membrane permeability. (Lipinski, Lombardo et al. 1997).

Binding ligands, that act as agonists, antagonists, or inverse agonistsand such that are known in the art can be diffused into or soaked withthe stabilized crystals of ERR-α to form a complex for collecting X-raydiffraction data. Alternatively, ligands known and unknown in the artcan be cocrystallized with ERR-α by mixing the ligand with ERR-α beforecrystallization.

To produce custom high affinity and very specific compounds, thestructure of ERR-α can be compared to the structure of a selectednon-targeted molecule and a hybrid constructed by changing the structureof residues at the binding site for a ligand for the residues at thesame positions of the non-target molecule. The process whereby thismodeling is achieved is referred to as homology structure modeling. Thisis done computationally by removing the side chains from the molecule ortarget of known structure and replacing them with the side chains of theunknown structure put in sterically plausible positions. In this way itcan be understood how the shapes of the active site cavities of thetargeted and non-targeted molecules differ. This process, therefore,provides information concerning how a bound ligand can be chemicallyaltered in order to produce compounds that will bind tightly andspecifically to the desired target but will simultaneously be stericallyprevented from binding to the non-targeted molecule. Likewise, knowledgeof portions of the bound ligands that are facing to the solvent wouldallow introduction of other functional groups for additionalpharmaceutical purposes. The use of homology structure modeling todesign ligands that bind more tightly to the target enzyme than to thenon-target enzyme has wide spread applicability.

Databases and Computer Systems

An amino acid sequence or nucleotide sequence of ERR-α and/or X-raydiffraction data, useful for computer molecular modeling of ERR-α or aportion thereof, can be provided in a variety of mediums to facilitateuse thereof. In one application of this embodiment, databases comprisingdata pertaining to X-ray diffraction data for a complex of ERR-α and aligand that forms a thioether bond with Cys325 of ERR-α, or at least oneERR-α subdomain thereof, is recorded on computer readable medium. Askilled artisan can readily appreciate how any of the presently knowncomputer readable media can be used to create a manufacture comprisingcomputer readable medium having recorded thereon data pertaining toX-ray diffraction data of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon an aminoacid sequence and/or atomic coordinate/X-ray diffraction data of thepresent invention. The choice of the data storage structure willgenerally be based on the means chosen to access the stored information.In addition, a variety of data processor programs and formats can beused to store the sequence and X-ray data information of the presentinvention on computer readable media. The sequence information can berepresented in a word processing text file, formatted incommercially-available software such as WordPerfect and MICROSOFT Word,or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. A skilled artisancan readily adapt any number of dataprocessor structuring formats (e.g.,text file or database) in order to obtain computer readable media havingrecorded thereon the information of the present invention.

By providing computer readable media having sequence and/or atomiccoordinates based on X-ray diffraction data, a skilled artisan canroutinely access the sequence and atomic coordinate or X-ray diffractiondata to model a related molecule, a subdomain, mimetic, or a ligandthereof. Computer algorithms are publicly and commercially availablewhich allow a skilled artisan to access this data provided in a computerreadable medium and analyze it for molecular modeling and/or RDD(rational drug design). See, e.g., (Mary Ann Liebert (Publishers) 1995).

The present invention further provides systems, particularlycomputer-based systems, which contain the sequence and/or diffractiondata described herein. Such systems are designed to do structuredetermination and RDD for ERR-α or at least one subdomain thereof.Non-limiting examples are microcomputer workstations available fromSilicon Graphics Incorporated and Sun Microsystems running UNIX based,Windows NT or IBM OS/2 operating systems.

A variety of comparing means can also be used to compare a targetsequence or target motif with the data storage means to identifystructural motifs or electron density maps derived in part from theatomic coordinate/X-ray diffraction data. A skilled artisan can readilyrecognize that any one of the publicly available computer modelingprograms can be used as the search means for the computer-based systemsof the present invention.

Integrated Procedures which Utilize the Present Invention

Molecular modeling is provided by the present invention for rationaldrug design (RDD) of mimetics and ligands that form a thioether bondwith Cys325 of ERR-α. As described above, the drug design paradigm usescomputer-modeling programs to determine potential mimetics and ligandswhich are expected to interact with sites on the protein. The potentialmimetics or ligands are then screened for activity and/or binding and/orinteraction. For ERR-α-related mimetics or ligands, screening methodscan be selected from assays for at least one biological activity ofERR-α.

Thus, the tools and methodologies provided by the present invention maybe used in procedures for identifying and designing ligands which bindin desirable ways with the target. Such procedures utilize an iterativeprocess whereby ligands are synthesized, tested and characterized. Newligands can be designed based on the information gained in the testingand characterization of the initial ligands and then such newlyidentified ligands can themselves be tested and characterized. Thisseries of processes may be repeated as many times as necessary to obtainligands with the desirable binding properties.

The following steps (1-7) serve as an example of the overall procedure:

-   -   1. A biological activity of a target is selected.    -   2. A ligand is identified that appears to be in some way        associated with the chosen biological activity (e.g., the ligand        may be an agonist, antagonist, or inverse agonist of a known        activity). The activity of the ligand may be tested by in vivo        and/or in vitro methods. A ligand of the present invention can        be, but is not limited to, at least one selected from a lipid, a        nucleic acid, a compound, a protein, an element, an antibody, a        saccharide, an isotope, a carbohydrate, an imaging agent, a        lipoprotein, a glycoprotein, an enzyme, a detectable probe, and        antibody or fragment thereof, or any combination thereof, which        can be detectably labeled as for labeling antibodies. Such        labels include, but are not limited to, enzymatic labels,        radioisotope or radioactive compounds or elements, fluorescent        compounds or metals, chemiluminescent compounds and        bioluminescent compounds. Alternatively, any other known        diagnostic or therapeutic agent can be used in a method of the        invention. Suitable compounds are then tested for activities in        relationship to the target. Complexes between ERR-α and ligands        are made either by co-crystallization or more commonly by        diffusing the ligand into the crystal. X-ray diffraction data        from the crystal complex are measured and a difference electron        density map is calculated. This process provides the precise        location of the bound ligand on the target molecule. The        difference Fourier is calculated using measure diffraction        amplitudes and the phases of these reflections calculated from        the coordinates.    -   3. Using the methods of the present invention, X-ray        crystallography is utilized to create electron density maps        and/or molecular models of the interaction of the ligand with        the target molecule. The entry of the coordinates of the target        into the computer programs discussed above results in the        calculation of most probable structure of the macromolecule.        These structures are combined and refined by additional        calculations using such programs to determine the probable or        actual three-dimensional structure of the target including        potential or actual active or binding sites of ligands. Such        molecular modeling (and related) programs useful for rational        drug design of ligands or mimetics are also provided by the        present invention.    -   4. The electron density maps and/or molecular models obtained in        Step 3 are compared to the electron density maps and/or        molecular models of a non-ligand containing target and the        observed/calculated differences are used to specifically locate        the binding of the ligand on the target or subunit.    -   5. Modeling tools, such as computational chemistry and computer        modeling, are used to adjust or modify the structure of the        ligand so that it can make additional or different interactions        with the target. The ligand design uses computer-modeling        programs which calculate how different molecules interact with        the various sites of the target, subunit, or a fragment thereof.        Thus, this procedure determines potential ligands or ligand        mimetics.    -   6. The newly designed ligand from Step 5 can be tested for its        biological activity using appropriate in vivo or in vitro tests,        including but not limited to the high-throughput screening        methods discussed above. The potential ligands or mimetics are        then screened for activity relating to ERR-α, or at least a        fragment thereof. Such screening methods are selected from        assays for at least one biological activity of the native        target. The resulting ligands or mimetics, provided by methods        of the present invention, are useful for treating, screening or        preventing diseases in animals, such as mammals (including        humans).    -   7. Of course, each of the above steps can be modified as desired        by those of skill in the art so as to refine the procedure for        the particular goal in mind. Also, additional X-ray diffraction        data may be collected on ERR-α, ERR-α/ligand complexes, ERR-α        structural target motifs and ERR-α subunit/ligand complexes at        any step or phase of the procedure. Such additional diffraction        data can be used to reconstruct electron density maps and        molecular models, which may further assist in the design and        selection of ligands with the desirable binding attributes.

It is to be understood that the present invention is considered toinclude stereoisomers as well as optical isomers, e.g., mixtures ofenantiomers as well as individual enantiomers and diastereomers, whicharise as a consequence of structural asymmetry in selected compounds,ligands or mimetics of the present series.

Some of the ligands disclosed or discovered by the methods herein maycontain one or more asymmetric centers and thus give rise toenantiomers, diastereomers, and other stereoisomeric forms. The presentinvention is also meant to encompass all such possible forms as well astheir racemic and resolved forms and mixtures thereof. When the ligandsdescribed or discovered herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless otherwise specified, it isintended to include both E and Z geometric isomers. All tautomers areintended to be encompassed by the present invention as well.

EXAMPLES

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the present invention andpractice the claimed methods. The following working examples therefore,specifically point out preferred embodiments of the present invention,and are not to be construed as limiting in any way the remainder of thedisclosure.

Protein Cloning, Expression and Purification

The ligand binding domain of human ERR-α (amino acids 290-519 ofSwiss-Prot P11474, SEQ ID NO:1) was subcloned into pDEST (Novagen) toproduce a construct with a PreScission protease cleavage site forcleavage of an included N-terminal His tag (SEQ ID NO:2). The plasmidwas co-transfected with linearized baculovirus DNA into Spodopterafrugiperda (Sf9) insect cells and the baculovirus was amplified andpurified from plaques from a titer 1−2×10⁸ pfu/mL. Sf9 cells weremaintained in ESF 921 media (Expression Systems. LLC, Woodland, Calif.)and grown in 2 L Erlenmeyer flasks at 27° C. The insect cells at a celldensity of 1.5×10⁶ cells/mL were infected with the baculovirus at amultiplicity of infection (MOI) of 1. Cells were harvested 3 dayspost-infection by centrifugation at 1200×g, rinsed with PBS supplementedwith protease inhibitors and stored at −80° C. until further use.Expression level of the protein was confirmed by Western blots usinganti-His antibody.

For purification, the cells containing recombinant human ERR-α weresuspended in 25 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 10 mM imidazole, 0.6 mMPMSF, 10 mM β-mercaptoethanol (buffer A) supplemented with CompleteProtease Inhibitor Cocktail (Roche). The suspension was sonicated usingBranson-450 sonicator and clarified by centrifugation at 100,000×g for 1hour. The supernatant was applied onto 5 ml Ni-NTA column (QIAGEN)pre-equilibrated in buffer A. The column was washed with 10 columnvolumes of 50 mM imidazole in buffer A, and developed by 250 mMimidazole in buffer A. The elution fractions containing ERR-α werepooled and dialyzed 0/N against 25 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mMdithiothreitol (buffer B). After dialysis, the protein was loaded onMonoQ 10/10 (GE Healthcare) and the column was developed by 50-1000 mMlinear gradient of NaCl in buffer B. The ERR-α was eluted at about 300mM NaCl and its purity was greater than 95% as judged by SDS-PAGE.Finally, the ERR-α was concentrated to 17 mg/ml at 2× molar access ofthe Compound 1, and delivered for crystallization in 25 mM Tris-HCl, pH8.0, 0.3 M NaCl, 5 mM dithiothreitol.

Crystallization and Data Collection

Using a hanging drop vapor diffusion method, crystals formed at 277° Kin a drop solution containing a 1:1 ratio of the solution of the ERR-αprotein and Compound 1 complex and a solution containing 1.4 M ammoniumsulfate, 100 mM Pipes pH 6.5 and 200 mM Na-thiocyanate. The drop wassuspended over a the same solution used to make the drop, the solutioncontaining 1.4 M ammonium sulfate, 100 mM Pipes pH 6.5 and 200 mMNa-thiocyanate.

The crystals were transferred to a cryoprotectant solution containing1.4 M ammonium sulfate, 100 mM Pipes pH 6.5, 200 mM Na-thiocyanate and25% glycerol. The crystals were then mounted and quickly frozen byimmersion in liquid nitrogen. X-ray diffraction data to a resolution of2.0 Å were collected on a Bruker AXS Proteum 6000 detector. Diffractiondata was indexed, integrated and scaled using the Proteum ProcessingProgram suite from Bruker AXS. Under these conditions, the crystalsbelong to the P6522 space group, with unit cell parameters a=b=103,c=110 Å, α=β=90 and γ=120. The structure was determined by molecularreplacement with CNX (Brunger, Adams et al. 1998) using the crystalstructure of ERR-α in complex with the peroxisome proliferator-activatedreceptor coactivator-1 (PGC1-α, pdb id 1XB7) as the search model(Kallen, Schlaeppi et al. 2004). Model building was done using theprogram O (Jones, Zou et al. 1991) and Coot (Emsley and Cowtan 2004).Refinement and map calculations were carried out using PHENIX (Adams,Grosse-Kunstleve et al. 2002). The final structure was refined to anRfactor of 21.1 and Rfree of 24.9. Inspection of the electron densitymap revealed that Compound 1 bound between helices 3 and 11 of ERR-α.

TABLE 1 Crystal and Refinement Parameters Parameter BPA Unit cell, Å a =b = 103.007 c = 110.017 Resolution, Å 2 Completeness, % 99.92Rmerge^(¥), %   10 (44.6) <I>/<_(σ1)> 8.20 (2.18) Rfactor^(#), % 21.1Rfreel^(¶), % 24.9 Values in parenthesis refer to the highest resolutionshell ^(¥)R_(merge) = Σ_(hkl) Σ₁ (|I₁ − <I>|/<I>), where I₁ is anindividual intensity measurement and <I> is the average intensity forthis reflection, with summation over all data. ^(#)Rfactor = Σ||F_(o)| −|F_(c)||/Σ|F_(o)|. ^(¶)10% of the total reflections withheld.

X-Ray Structure Discussion

A comparison between the crystal structure of the complex of ERR-α andCompound 1 and the previously determined crystal structure of thecomplex of ERR-α and PGC1-α showed conserved arrangement of secondarystructural elements throughout most of the structure (FIG. 1A). Theexcellent data quality obtained from the complex of ERR-α and Compound 1allowed unambiguous placement of Compound 1 in the electron density(FIG. 2A). The positions of the sulfurs of Cys325 and the thiazole ofCompound 1 were validated by the calculation of a sulfur anomalous map(FIG. 2B). Anomalous signal from all other cysteines and methioninesthroughout the protein were also observed. Secondary structurerearrangement upon Compound 1 binding included the displacement of helix11 and helix 12. The side chains of residues F328 and F495 must move toaccommodate the ligand as shown on FIG. 1B. The structure of the complexof ERR-α and Compound 1 also showed helix 12 occupying the position ofthe activation peptide. Some shifting was also observed on helix 3 andits side chains to make room for Compound 1 and maximize itsinteractions with helix 12.

ERR-α Covalent Modification

Inspection of the binding site and protein-ligand interactions revealedCompound 1 binding covalently to the protein by a thioether bond betweenthe carbon C5 of the Compound 1 and the sulfur from Cys325. The distancebetween the sulfur of Cys325 and carbon C6 is 2.44 Å, consistent with acovalent bond. The double bond between C6 and C7 of the Compound 1 mustreduce in order to allow C6 to covalently link the protein. Ameasurement of this distance is 1.55 Å, consistent with a carbon-carbonsingle bond distance and evidence that covalent modification occurred.

Other Interactions Observed and to be Explored

The hydrophobic pocket of ERR-α presents a few polar groups withinH-bond distance to Compound 1 that could be explored in furtheroptimization to develop additional ligands. These residues include E331,R372, F382-carbonyl and L324-carbonyl (Table 2). The carbonyl of F382hydrogen bonds (3.41 Å) with N2 in the cyano. The carbons C13 and C14 ofthe 3-Trifluoromethyl-benzonitrile are close enough to the carbonyl ofL324 that an H-donor substitution would take advantage of the proximityto this residue. The cyano group of Compound 1 is 3.2 Åfrom R372,another proton donor. Substituting the cyano with an electronegativeatom could potentially optimize this part of the molecule.

TABLE 2 List of contacts between ERR-α and Compound 1 within a distanceof 3.5 Å

Source atoms target atoms distance Å Leu 324A CB . . . Lig 1I O02 . . .3.42 Leu 324A O . . . Lig 1I C19 . . . 3.28 Cys 325A CB . . . Lig 1I C06. . . 3.36 Cys 325A SG . . . Lig 1I C07 . . . 2.94 . . . Lig 1I C12 . .. 3.48 . . . Lig 1I C05 . . . 3.15 . . . Lig 1I C06 . . . 2.44 . . . Lig1I C04 . . . 3.24 Phe 328A CG . . . Lig 1I C14 . . . 3.40 . . . Lig 1IC15 . . . 3.17 Phe 328A CD1 . . . Lig 1I C14 . . . 3.39 Phe 328A CD2 . .. Lig 1I C15 . . . 3.33 Glu 331A CG . . . Lig 1I N23 . . . 3.29 Arg 372ANH2 . . . Lig 1I N23 . . . 3.21*** Phe 382A O . . . Lig 1I N23 . . .3.41* Ala 396A O . . . Lig 1I C01 . . . 3.27 Leu 398A CD2 . . . Lig 1IO02 . . . 3.28 . . . Lig 1I C01 . . . 3.40 Val 491A CG1 . . . Lig 1I F27. . . 3.19 Phe 495A CZ . . . Lig 1I C03 . . . 3.33 . . . Lig 1I C16 . .. 3.19 . . . Lig 1I O17 . . . 3.33 Met 506A CE . . . Lig 1I C14 . . .3.40 . . . Lig 1I C15 . . . 3.30 ***indicates strong hydrogen bonds

LC/MS Experiments

To monitor the kinetics of association and dissociation for ligands thatformed a complex with ERR-α by forming a covalent bond to Cys325 ofERR-α, LC/MS detection was employed. LC analysis was performed on anAgilent 1100 LC system that was in line with an Agilent MSD TOF for massdetection. Software provided by the vendor was used to deconvolute theESI positive ion quadrupole time-of-flight spectra. To measureassociation rates, typically a 1 μM ERR-α solution was mixed with anequal volume of a 2 μM ligand solution. 20 uL aliquots of the mixedsolutions were removed at time zero and at regular defined timeintervals and diluted out in 100 μL of a 0.1% TFA, 10% CH₃CN solution toquench the reaction. 50 μL samples of the diluted analyte were processedon the LC/MS instrument. Similarly, for determination of thedissociation rates, the mixed solutions of ERR-α and the ligand werefirst equilibrated up to one hour to allow formation of the complex. Attime 0, a 20-fold excess of a competing ligand (Compound 2) was addedand aliquots were removed and processed as described for the associationrate experiments. Final concentrations for the ligand, ERR-α, and thecompeting ligand were 1 μM, 0.5 μM, and 20 μM, respectively. Allexperiments were carried out in buffer containing 25 mM HEPES, pH 7.9,200 mM KCl and 3% DMSO at 37° C.

For the apo form of ERR-α, a mass of 27042 was detected, correspondingto an amino acid sequence where the initiator methionine is cleavedfollowed by N-terminal acetylation. This is the “Expected Mass” shown inTable 3. When the ERR-α protein was incubated with a ligand that formeda covalent bond to Cys325 of ERR-α, there was a time dependent increasein the mass of the protein equal to the mass of the compound+1 amu(Table 3).

TABLE 3 Observed mass changes for 2 ligands that formed a complex withERR-α Expected Observed Mass Ligand Compound Mass Mass Difference MassCompound Structure Number (daltons) (daltons) (daltons) (daltons)

Compound 1 27042 27461 419 420.0392

Compound 2 27042 27494 452 453.0494

The progress of the reaction was expressed as a fraction of theintegrated areas observed for the expected mass (27042 amu) and observedmass (27042+compound amu−1) with the following expression:

${{progress}\mspace{14mu} {of}\mspace{14mu} {reaction}} = \frac{{area}\mspace{14mu} {of}\mspace{14mu} {observed}\mspace{14mu} {mass}}{\begin{matrix}{{{area}\mspace{14mu} {of}\mspace{14mu} {observed}\mspace{14mu} {mass}} +} \\{{area}\mspace{14mu} {of}\mspace{14mu} {expected}\mspace{14mu} {mass}}\end{matrix}}$

Similarly for the experiments to measure dissociation rates, incubationof a complex of ERR-α and a ligand with the competing ligand resulted ina time dependent change in the mass of the complex of ERR-α and theligand, equal to the mass difference of the ligand and the competingligand. To standardize the experiments to measure dissociation rates fordifferent ligands, the same competing ligand (Compound 2) was used inall experiments. The progress of the reaction was also expressed as afraction of the area observed for the complex of ERR-α and the competingligand over the sum of total area for the complex of ERR-α with theligand and the complex of ERR-α with the competing ligand.

Association and dissociation rates for ligands were determined byfitting fractional values obtained for the progress of the reaction as afunction of time using a single exponential equation

progress of reaction=e^(−kt)

where k is the apparent rate constant. For the dissociation experiments,the reaction is assumed to be first order and dissociation rates, k_(d),are expressed as s⁻¹. For the association rates, k^(a), the reaction isassumed to be second order and the rates are expressed as M⁻¹ s⁻¹ bydividing the apparent rate constants by the concentration of the proteindetermined by experimental conditions (Table 4). Apparent half-lives(t_(0.5)) were calculated using the equation:

$t_{0.5} = \frac{0.693}{kd}$

TABLE 4 Kinetic rate constants for ligands that formed a complex withERR-α Dissociation rates determined with 20-fold excess Compound 2 as acompeting ligand Compound k_(a) k_(d) Compound Structure Number M⁻¹ s⁻¹s⁻¹

Compound 1 6600 1.10 × 10⁻⁵

Compound 3 Not Determined 1.53 × 10⁻⁵

Compound 4 Not Determined 2.19 × 10⁻⁵

Compound 5 Not Determined 2.05 × 10⁻⁵

TR-FRET Assay

Time-resolved Fluorescence resonance energy transfer (TR-FRET)experiments were performed to examine the functional activity of theERR-α ligands. The components of this homogeneous secondary assayincluded: the purified ERR-α protein (SEQ ID NO:2), a GST-labeled-hSRC2co-activator polypeptide, and a fluorescent donor/acceptor pair from CISbio international htrf/bioassays (Bedford, Mass.) using both an α-GSTEuropium Cryptate (Eu) label and an α⁶His-XL665 (allophycocyanin)fluorophore.

For TR-FRET measurements, the reaction was buffered in 25 mM Tris pH 8,2.5 mM Hepes, 20 mM KCl, 1 mM DTT, and 0.05 mg/mL BSA (-lipids). Thefinal concentrations of reagents were 6 nM of ERR-α protein, 6 nMGST-SRC-2 peptide, 30 nM Eu cryptate, and 7.5 nM XL665. Reactions wereallowed to reach equilibrium at 25° C. for 4-18 hours before collectingdata on the Analyst from LJL Biosystems (Molecular Devices Sunnyvale,Calif.). As a time-resolved method, the samples were excited at 340 nMand emission was collected for 1 ms at both 615 and 665 nm with delaysof 400 and 75 μs, respectively. Dose response curves were fitted using ahyperbolic equation and the data reported in Table 5 is the average ofthree independent experiments.

TABLE 5 EC50 values determined by TR-FRET measurements Compound CompoundStructure Number EC50 (nM)

Compound 1 54

Compound 2 11

TABLE 6 Coordinates for crystal structure of the complex of ERR-α andCompound 1 CRYST1 103.007 103.007 110.017 90.00 90.00 120.00 P 65 2 2SCALE1 0.009708 0.005605 0.000000 0.00000 SCALE2 0.000000 0.0112100.000000 0.00000 SCALE3 0.000000 0.000000 0.009090 0.00000 ATOM 1 CB HISA 280 22.940 21.391 17.566 1.00 25.58 A C ATOM 2 CG HIS A 280 21.54421.696 17.118 1.00 23.84 A C ATOM 3 CD2 HIS A 280 20.344 21.364 17.6481.00 24.17 A C ATOM 4 ND1 HIS A 280 21.271 22.448 15.990 1.00 28.34 A NATOM 5 CE1 HIS A 280 19.963 22.557 15.845 1.00 27.40 A C ATOM 6 NE2 HISA 280 19.375 21.909 16.837 1.00 27.33 A N ATOM 7 C HIS A 280 25.28920.755 16.965 1.00 21.27 A C ATOM 8 O HIS A 280 25.430 19.936 17.8721.00 17.88 A O ATOM 9 N HIS A 280 23.397 19.993 15.595 1.00 34.65 A NATOM 10 CA HIS A 280 23.905 21.085 16.419 1.00 25.98 A C ATOM 11 N HIS A281 26.308 21.409 16.420 1.00 19.22 A N ATOM 12 CA HIS A 281 27.68521.080 16.764 1.00 19.90 A C ATOM 13 CB HIS A 281 28.556 21.123 15.5181.00 18.01 A C ATOM 14 CG HIS A 281 28.150 20.134 14.477 1.00 24.32 A CATOM 15 CD2 HIS A 281 27.629 18.889 14.587 1.00 25.01 A C ATOM 16 ND1HIS A 281 28.249 20.389 13.125 1.00 31.11 A N ATOM 17 CE1 HIS A 28127.819 19.338 12.450 1.00 27.61 A C ATOM 18 NE2 HIS A 281 27.440 18.41413.314 1.00 27.91 A N ATOM 19 C HIS A 281 28.279 21.970 17.855 1.0022.70 A C ATOM 20 O HIS A 281 29.413 21.744 18.294 1.00 13.09 A O ATOM21 N LEU A 282 27.514 22.980 18.270 1.00 16.02 A N ATOM 22 CA LEU A 28227.905 23.856 19.367 1.00 14.99 A C ATOM 23 CB LEU A 282 28.083 23.04520.650 1.00 15.69 A C ATOM 24 CG LEU A 282 26.922 22.138 21.057 1.0016.92 A C ATOM 25 CD1 LEU A 282 27.340 21.212 22.192 1.00 20.99 A C ATOM26 CD2 LEU A 282 25.730 22.971 21.460 1.00 19.45 A C ATOM 27 C LEU A 28229.184 24.642 19.076 1.00 15.48 A C ATOM 28 O LEU A 282 29.837 25.13919.998 1.00 13.42 A O ATOM 29 N GLU A 283 29.537 24.772 17.801 1.0011.87 A N ATOM 30 CA GLU A 283 30.759 25.485 17.443 1.00 13.95 A C ATOM31 CB GLU A 283 31.047 25.331 15.956 1.00 20.90 A C ATOM 32 CG GLU A 28331.088 23.869 15.523 1.00 23.92 A C ATOM 33 CD GLU A 283 32.328 23.53714.729 1.00 28.45 A C ATOM 34 OE1 GLU A 283 33.070 22.625 15.149 1.0035.17 A O ATOM 35 OE2 GLU A 283 32.570 24.191 13.693 1.00 36.58 A O ATOM36 C GLU A 283 30.776 26.966 17.847 1.00 14.78 A C ATOM 37 O GLU A 28331.844 27.555 18.007 1.00 11.19 A O ATOM 38 N VAL A 284 29.606 27.56818.022 1.00 12.37 A N ATOM 39 CA VAL A 284 29.553 28.968 18.444 1.0014.42 A C ATOM 40 CB VAL A 284 28.104 29.518 18.479 1.00 16.39 A C ATOM41 CG1 VAL A 284 27.339 28.954 19.674 1.00 12.80 A C ATOM 42 CG2 VAL A284 28.125 31.046 18.510 1.00 14.14 A C ATOM 43 C VAL A 284 30.24529.204 19.801 1.00 16.08 A C ATOM 44 O VAL A 284 30.691 30.316 20.0901.00 16.22 A O ATOM 45 N LEU A 285 30.350 28.164 20.626 1.00 12.98 A NATOM 46 CA LEU A 285 31.010 28.301 21.924 1.00 13.92 A C ATOM 47 CB LEUA 285 30.747 27.086 22.808 1.00 12.78 A C ATOM 48 CG LEU A 285 29.31626.963 23.325 1.00 13.18 A C ATOM 49 CD1 LEU A 285 29.133 25.636 24.0371.00 11.18 A C ATOM 50 CD2 LEU A 285 28.979 28.122 24.237 1.00 14.27 A CATOM 51 C LEU A 285 32.516 28.566 21.806 1.00 14.98 A C ATOM 52 O LEU A285 33.149 29.045 22.749 1.00 14.98 A O ATOM 53 N PHE A 286 33.08628.265 20.644 1.00 16.74 A N ATOM 54 CA PHE A 286 34.484 28.594 20.3891.00 11.61 A C ATOM 55 CB PHE A 286 35.096 27.627 19.366 1.00 11.98 A CATOM 56 CG PHE A 286 35.234 26.203 19.868 1.00 11.57 A C ATOM 57 CD1 PHEA 286 34.572 25.160 19.235 1.00 10.35 A C ATOM 58 CD2 PHE A 286 36.02025.913 20.969 1.00 11.94 A C ATOM 59 CE1 PHE A 286 34.695 23.845 19.6931.00 9.95 A C ATOM 60 CE2 PHE A 286 36.156 24.598 21.430 1.00 9.44 A CATOM 61 CZ PHE A 286 35.486 23.568 20.788 1.00 8.04 A C ATOM 62 C PHE A286 34.663 30.055 19.935 1.00 17.58 A C ATOM 63 O PHE A 286 35.77430.580 19.949 1.00 15.23 A O ATOM 64 N GLN A 287 33.570 30.719 19.5611.00 18.72 A N ATOM 65 CA GLN A 287 33.680 32.084 19.048 1.00 23.71 A CATOM 66 CB GLN A 287 32.472 32.466 18.178 1.00 25.11 A C ATOM 67 CG GLNA 287 32.869 33.179 16.866 1.00 39.90 A C ATOM 68 CD GLN A 287 32.06434.458 16.570 1.00 40.35 A C ATOM 69 OE1 GLN A 287 30.893 34.587 16.9421.00 28.44 A O ATOM 70 NE2 GLN A 287 32.704 35.408 15.892 1.00 40.81 A NATOM 71 C GLN A 287 33.869 33.116 20.152 1.00 25.99 A C ATOM 72 O GLN A287 34.713 34.007 20.048 1.00 34.52 A O ATOM 73 N GLY A 288 33.08733.015 21.213 1.00 18.89 A N ATOM 74 CA GLY A 288 33.075 34.089 22.1911.00 33.22 A C ATOM 75 C GLY A 288 34.224 34.056 23.181 1.00 35.28 A CATOM 76 O GLY A 288 35.141 33.240 23.057 1.00 32.12 A O ATOM 77 N PRO A289 34.210 34.983 24.149 1.00 34.30 A N ATOM 78 CD PRO A 289 33.79636.396 24.046 1.00 29.11 A C ATOM 79 CA PRO A 289 34.948 34.726 25.3861.00 27.66 A C ATOM 80 CB PRO A 289 34.369 35.771 26.335 1.00 28.11 A CATOM 81 CG PRO A 289 34.161 36.971 25.414 1.00 31.29 A C ATOM 82 C PRO A289 34.675 33.299 25.881 1.00 22.34 A C ATOM 83 O PRO A 289 33.58232.759 25.687 1.00 15.25 A O ATOM 84 N VAL A 290 35.679 32.702 26.5101.00 19.05 A N ATOM 85 CA VAL A 290 35.700 31.267 26.788 1.00 15.83 A CATOM 86 CB VAL A 290 37.109 30.846 27.208 1.00 11.51 A C ATOM 87 CG1 VALA 290 37.480 31.530 28.509 1.00 13.50 A C ATOM 88 CG2 VAL A 290 37.20729.330 27.339 1.00 13.64 A C ATOM 89 C VAL A 290 34.728 30.851 27.8911.00 15.39 A C ATOM 90 O VAL A 290 34.453 29.662 28.101 1.00 11.02 A OATOM 91 N ASN A 291 34.196 31.834 28.600 1.00 13.40 A N ATOM 92 CA ASN A291 33.413 31.533 29.789 1.00 14.32 A C ATOM 93 CB ASN A 291 33.29432.797 30.618 1.00 25.98 A C ATOM 94 CG ASN A 291 34.657 33.480 30.7861.00 42.18 A C ATOM 95 OD1 ASN A 291 35.463 33.076 31.641 1.00 33.92 A OATOM 96 ND2 ASN A 291 34.949 34.469 29.922 1.00 23.39 A N ATOM 97 C ASNA 291 32.080 30.835 29.497 1.00 14.00 A C ATOM 98 O ASN A 291 31.57330.069 30.323 1.00 13.69 A O ATOM 99 N ALA A 292 31.541 31.058 28.3021.00 12.59 A N ATOM 100 CA ALA A 292 30.354 30.331 27.867 1.00 12.77 A CATOM 101 CB ALA A 292 29.745 30.981 26.628 1.00 12.55 A C ATOM 102 C ALAA 292 30.698 28.860 27.597 1.00 12.45 A C ATOM 103 O ALA A 292 29.90927.967 27.891 1.00 10.66 A O ATOM 104 N LEU A 293 31.875 28.618 27.0241.00 12.65 A N ATOM 105 CA LEU A 293 32.311 27.256 26.729 1.00 11.33 A CATOM 106 CB LEU A 293 33.588 27.260 25.876 1.00 7.25 A C ATOM 107 CG LEUA 293 34.298 25.897 25.723 1.00 9.88 A C ATOM 108 CD1 LEU A 293 33.35224.814 25.182 1.00 9.87 A C ATOM 109 CD2 LEU A 293 35.543 26.019 24.8471.00 10.09 A C ATOM 110 C LEU A 293 32.538 26.507 28.037 1.00 8.89 A CATOM 111 O LEU A 293 32.065 25.387 28.214 1.00 9.23 A O ATOM 112 N VAL A294 33.258 27.147 28.953 1.00 8.15 A N ATOM 113 CA VAL A 294 33.52926.570 30.262 1.00 12.41 A C ATOM 114 CB VAL A 294 34.406 27.505 31.1261.00 10.83 A C ATOM 115 CG1 VAL A 294 34.342 27.088 32.582 1.00 11.13 AC ATOM 116 CG2 VAL A 294 35.843 27.502 30.623 1.00 8.98 A C ATOM 117 CVAL A 294 32.234 26.263 31.014 1.00 12.48 A C ATOM 118 O VAL A 29432.106 25.209 31.622 1.00 10.18 A O ATOM 119 N SER A 295 31.284 27.19430.981 1.00 10.91 A N ATOM 120 CA SER A 295 29.997 26.974 31.625 1.009.45 A C ATOM 121 CB SER A 295 29.096 28.212 31.499 1.00 14.43 A C ATOM122 OG SER A 295 27.744 27.897 31.806 1.00 20.43 A O ATOM 123 C SER A295 29.325 25.750 31.023 1.00 12.36 A C ATOM 124 O SER A 295 28.73624.939 31.739 1.00 10.39 A O ATOM 125 N HIS A 296 29.425 25.605 29.7061.00 11.75 A N ATOM 126 CA HIS A 296 28.855 24.439 29.035 1.00 10.68 A CATOM 127 CB HIS A 296 28.941 24.582 27.517 1.00 9.76 A C ATOM 128 CG HISA 296 28.506 23.361 26.772 1.00 10.90 A C ATOM 129 CD2 HIS A 296 29.18722.254 26.392 1.00 10.88 A C ATOM 130 ND1 HIS A 296 27.209 23.180 26.3261.00 12.76 A N ATOM 131 CE1 HIS A 296 27.118 22.020 25.703 1.00 14.01 AC ATOM 132 NE2 HIS A 296 28.304 21.437 25.726 1.00 14.24 A N ATOM 133 CHIS A 296 29.523 23.132 29.476 1.00 11.92 A C ATOM 134 O HIS A 29628.847 22.141 29.761 1.00 11.60 A O ATOM 135 N LEU A 297 30.847 23.12029.531 1.00 9.35 A N ATOM 136 CA LEU A 297 31.553 21.917 29.972 1.0010.01 A C ATOM 137 CB LEU A 297 33.061 22.123 29.911 1.00 9.01 A C ATOM138 CG LEU A 297 33.553 22.379 28.495 1.00 7.69 A C ATOM 139 CD1 LEU A297 35.024 22.708 28.538 1.00 5.55 A C ATOM 140 CD2 LEU A 297 33.24621.150 27.637 1.00 6.97 A C ATOM 141 C LEU A 297 31.169 21.520 31.3841.00 10.36 A C ATOM 142 O LEU A 297 31.045 20.336 31.691 1.00 10.64 A OATOM 143 N LEU A 298 30.994 22.513 32.251 1.00 10.74 A N ATOM 144 CA LEUA 298 30.614 22.245 33.629 1.00 12.52 A C ATOM 145 CB LEU A 298 30.65823.524 34.468 1.00 10.34 A C ATOM 146 CG LEU A 298 32.041 24.076 34.8151.00 12.08 A C ATOM 147 CD1 LEU A 298 31.920 25.418 35.555 1.00 8.73 A CATOM 148 CD2 LEU A 298 32.841 23.076 35.640 1.00 9.28 A C ATOM 149 C LEUA 298 29.232 21.588 33.682 1.00 10.48 A C ATOM 150 O LEU A 298 29.01820.646 34.450 1.00 10.70 A O ATOM 151 N VAL A 299 28.311 22.066 32.8491.00 10.43 A N ATOM 152 CA VAL A 299 26.954 21.499 32.789 1.00 12.43 A CATOM 153 CB VAL A 299 26.000 22.363 31.927 1.00 15.42 A C ATOM 154 CG1VAL A 299 24.746 21.569 31.528 1.00 11.39 A C ATOM 155 CG2 VAL A 29925.613 23.630 32.668 1.00 16.30 A C ATOM 156 C VAL A 299 26.914 20.04632.293 1.00 12.64 A C ATOM 157 O VAL A 299 26.206 19.223 32.852 1.0011.54 A O ATOM 158 N VAL A 300 27.670 19.734 31.246 1.00 13.30 A N ATOM159 CA VAL A 300 27.618 18.398 30.646 1.00 13.01 A C ATOM 160 CB VAL A300 28.052 18.407 29.165 1.00 13.09 A C ATOM 161 CG1 VAL A 300 27.19819.369 28.359 1.00 13.58 A C ATOM 162 CG2 VAL A 300 29.516 18.770 29.0501.00 11.79 A C ATOM 163 C VAL A 300 28.481 17.387 31.407 1.00 14.75 A CATOM 164 O VAL A 300 28.515 16.209 31.069 1.00 12.75 A O ATOM 165 N GLUA 301 29.191 17.861 32.422 1.00 14.54 A N ATOM 166 CA GLU A 301 29.90116.974 33.325 1.00 15.49 A C ATOM 167 CB GLU A 301 30.595 17.793 34.4101.00 13.73 A C ATOM 168 CG GLU A 301 31.478 17.003 35.356 1.00 18.04 A CATOM 169 CD GLU A 301 32.572 16.200 34.658 1.00 14.78 A C ATOM 170 OE1GLU A 301 33.082 15.260 35.292 1.00 16.40 A O ATOM 171 OE2 GLU A 30132.929 16.494 33.496 1.00 13.16 A O ATOM 172 C GLU A 301 28.886 16.00433.931 1.00 19.75 A C ATOM 173 O GLU A 301 27.857 16.426 34.455 1.0020.35 A O ATOM 174 N PRO A 302 29.169 14.698 33.850 1.00 17.66 A N ATOM175 CD PRO A 302 30.417 14.127 33.304 1.00 14.23 A C ATOM 176 CA PRO A302 28.245 13.671 34.349 1.00 19.35 A C ATOM 177 CB PRO A 302 29.05912.380 34.237 1.00 19.40 A C ATOM 178 CG PRO A 302 30.105 12.673 33.1781.00 19.78 A C ATOM 179 C PRO A 302 27.834 13.888 35.801 1.00 19.84 A CATOM 180 O PRO A 302 28.633 14.369 36.608 1.00 18.01 A O ATOM 181 N GLUA 303 26.595 13.527 36.129 1.00 24.87 A N ATOM 182 CA GLU A 303 26.12713.587 37.513 1.00 34.74 A C ATOM 183 CB GLU A 303 24.637 13.246 37.6011.00 31.77 A C ATOM 184 CG GLU A 303 23.731 14.164 36.791 1.00 46.93 A CATOM 185 CD GLU A 303 23.742 15.601 37.287 1.00 48.48 A C ATOM 186 OE1GLU A 303 24.025 15.820 38.486 1.00 53.83 A O ATOM 187 OE2 GLU A 30323.459 16.510 36.475 1.00 51.75 A O ATOM 188 C GLU A 303 26.913 12.59438.359 1.00 28.66 A C ATOM 189 O GLU A 303 27.459 11.623 37.837 1.0018.80 A O ATOM 190 N LYS A 304 26.968 12.830 39.663 1.00 27.38 A N ATOM191 CA LYS A 304 27.639 11.894 40.552 1.00 28.05 A C ATOM 192 CB LYS A304 27.673 12.425 41.986 1.00 30.61 A C ATOM 193 CG LYS A 304 27.34013.898 42.124 1.00 41.26 A C ATOM 194 CD LYS A 304 28.151 14.772 41.1741.00 46.02 A C ATOM 195 CE LYS A 304 27.391 16.062 40.869 1.00 50.74 A CATOM 196 NZ LYS A 304 25.928 15.791 40.674 1.00 44.82 A N ATOM 197 C LYSA 304 26.906 10.554 40.493 1.00 26.57 A C ATOM 198 O LYS A 304 25.67810.508 40.464 1.00 26.65 A O ATOM 199 N LEU A 305 27.657 9.462 40.4421.00 24.73 A N ATOM 200 CA LEU A 305 27.040 8.143 40.446 1.00 23.98 A CATOM 201 CB LEU A 305 27.729 7.207 39.453 1.00 25.79 A C ATOM 202 CG LEUA 305 27.617 7.530 37.961 1.00 26.42 A C ATOM 203 CD1 LEU A 305 28.1926.385 37.146 1.00 21.98 A C ATOM 204 CD2 LEU A 305 26.175 7.793 37.5681.00 27.99 A C ATOM 205 C LEU A 305 27.099 7.542 41.838 1.00 23.16 A CATOM 206 O LEU A 305 28.014 7.834 42.616 1.00 23.56 A O ATOM 207 N TYR A306 26.110 6.713 42.151 1.00 22.53 A N ATOM 208 CA TYR A 306 26.1435.911 43.365 1.00 26.89 A C ATOM 209 CB TYR A 306 24.880 6.128 44.1981.00 27.79 A C ATOM 210 CG TYR A 306 24.844 7.508 44.794 1.00 31.63 A CATOM 211 CD1 TYR A 306 24.342 8.585 44.071 1.00 29.49 A C ATOM 212 CE1TYR A 306 24.334 9.854 44.603 1.00 32.64 A C ATOM 213 CD2 TYR A 30625.356 7.746 46.061 1.00 33.55 A C ATOM 214 CE2 TYR A 306 25.351 9.00846.604 1.00 36.28 A C ATOM 215 CZ TYR A 306 24.841 10.060 45.874 1.0042.00 A C ATOM 216 OH TYR A 306 24.839 11.320 46.426 1.00 52.73 A O ATOM217 C TYR A 306 26.320 4.448 42.998 1.00 26.38 A C ATOM 218 O TYR A 30625.511 3.882 42.274 1.00 22.90 A O ATOM 219 N ALA A 307 27.398 3.85043.488 1.00 23.02 A N ATOM 220 CA ALA A 307 27.699 2.460 43.198 1.0024.52 A C ATOM 221 CB ALA A 307 29.188 2.218 43.325 1.00 22.08 A C ATOM222 C ALA A 307 26.920 1.488 44.091 1.00 27.85 A C ATOM 223 O ALA A 30726.705 0.338 43.719 1.00 27.89 A O ATOM 224 N MET A 308 26.505 1.94545.267 1.00 29.84 A N ATOM 225 CA MET A 308 25.797 1.076 46.208 1.0039.93 A C ATOM 226 CB MET A 308 26.185 1.413 47.654 1.00 33.82 A C ATOM227 CG MET A 308 27.682 1.467 47.915 1.00 34.50 A C ATOM 228 SD MET A308 28.540 −0.093 47.616 1.00 42.64 A S ATOM 229 CE MET A 308 30.2210.421 47.935 1.00 45.56 A C ATOM 230 C MET A 308 24.274 1.148 46.0591.00 39.26 A C ATOM 231 O MET A 308 23.726 2.189 45.691 1.00 44.59 A OATOM 232 N PRO A 309 23.589 0.030 46.348 1.00 50.14 A N ATOM 233 CD PROA 309 24.235 −1.277 46.579 1.00 48.34 A C ATOM 234 CA PRO A 309 22.124−0.058 46.414 1.00 47.55 A C ATOM 235 CB PRO A 309 21.898 −1.375 47.1551.00 49.88 A C ATOM 236 CG PRO A 309 23.069 −2.217 46.766 1.00 58.07 A CATOM 237 C PRO A 309 21.516 1.091 47.217 1.00 52.44 A C ATOM 238 O PRO A309 21.952 1.339 48.347 1.00 50.51 A O ATOM 239 N LEU A 317 25.154−4.678 48.314 1.00 52.79 A N ATOM 240 CA LEU A 317 25.358 −5.964 48.9771.00 51.70 A C ATOM 241 CB LEU A 317 24.020 −6.568 49.438 1.00 56.53 A CATOM 242 CG LEU A 317 22.702 −6.226 48.722 1.00 59.14 A C ATOM 243 CD1LEU A 317 22.266 −4.787 49.001 1.00 58.56 A C ATOM 244 CD2 LEU A 31722.757 −6.501 47.218 1.00 52.71 A C ATOM 245 C LEU A 317 26.145 −6.95548.110 1.00 44.57 A C ATOM 246 O LEU A 317 26.440 −8.073 48.538 1.0045.10 A O ATOM 247 N LEU A 318 26.484 −6.532 46.895 1.00 41.57 A N ATOM248 CA LEU A 318 27.298 −7.340 45.993 1.00 31.89 A C ATOM 249 CB LEU A318 27.230 −6.782 44.573 1.00 29.81 A C ATOM 250 CG LEU A 318 25.911−6.909 43.816 1.00 32.72 A C ATOM 251 CD1 LEU A 318 25.977 −6.147 42.5031.00 26.20 A C ATOM 252 CD2 LEU A 318 25.595 −8.371 43.573 1.00 35.10 AC ATOM 253 C LEU A 318 28.749 −7.350 46.456 1.00 26.04 A C ATOM 254 OLEU A 318 29.186 −6.435 47.152 1.00 22.04 A O ATOM 255 N PRO A 31929.503 −8.394 46.079 1.00 23.87 A N ATOM 256 CD PRO A 319 29.055 −9.61045.383 1.00 21.16 A C ATOM 257 CA PRO A 319 30.942 −8.415 46.362 1.0018.87 A C ATOM 258 CB PRO A 319 31.398 −9.751 45.765 1.00 18.38 A C ATOM259 CG PRO A 319 30.160 −10.570 45.651 1.00 20.01 A C ATOM 260 C PRO A319 31.641 −7.244 45.651 1.00 17.27 A C ATOM 261 O PRO A 319 31.106−6.692 44.686 1.00 16.55 A O ATOM 262 N ALA A 320 32.823 −6.875 46.1301.00 17.00 A N ATOM 263 CA ALA A 320 33.549 −5.723 45.601 1.00 19.01 A CATOM 264 CB ALA A 320 34.920 −5.614 46.257 1.00 16.56 A C ATOM 265 C ALAA 320 33.690 −5.741 44.081 1.00 16.68 A C ATOM 266 O ALA A 320 33.388−4.749 43.415 1.00 15.58 A O ATOM 267 N VAL A 321 34.157 −6.860 43.5361.00 13.60 A N ATOM 268 CA VAL A 321 34.378 −6.951 42.093 1.00 16.13 A CATOM 269 CB VAL A 321 34.927 −8.323 41.674 1.00 15.02 A C ATOM 270 CG1VAL A 321 35.190 −8.336 40.180 1.00 15.96 A C ATOM 271 CG2 VAL A 32136.197 −8.633 42.435 1.00 22.36 A C ATOM 272 C VAL A 321 33.112 −6.67041.287 1.00 16.45 A C ATOM 273 O VAL A 321 33.146 −5.947 40.287 1.0015.37 A O ATOM 274 N ALA A 322 31.997 −7.243 41.732 1.00 15.98 A N ATOM275 CA ALA A 322 30.734 −7.140 41.010 1.00 14.85 A C ATOM 276 CB ALA A322 29.700 −8.085 41.622 1.00 15.22 A C ATOM 277 C ALA A 322 30.213−5.708 41.008 1.00 15.26 A C ATOM 278 O ALA A 322 29.718 −5.212 40.0021.00 15.54 A O ATOM 279 N THR A 323 30.327 −5.059 42.154 1.00 15.80 A NATOM 280 CA THR A 323 29.934 −3.669 42.309 1.00 14.87 A C ATOM 281 CBTHR A 323 30.219 −3.211 43.745 1.00 14.02 A C ATOM 282 OG1 THR A 32329.453 −4.027 44.643 1.00 16.97 A O ATOM 283 CG2 THR A 323 29.872 −1.73443.946 1.00 16.94 A C ATOM 284 C THR A 323 30.703 −2.799 41.326 1.0014.96 A C ATOM 285 O THR A 323 30.121 −1.995 40.605 1.00 15.71 A O ATOM286 N LEU A 324 32.015 −2.983 41.281 1.00 14.81 A N ATOM 287 CA LEU A324 32.862 −2.206 40.385 1.00 13.95 A C ATOM 288 CB LEU A 324 34.334−2.495 40.671 1.00 11.37 A C ATOM 289 CG LEU A 324 34.922 −1.806 41.8991.00 8.98 A C ATOM 290 CD1 LEU A 324 36.314 −2.336 42.199 1.00 13.92 A CATOM 291 CD2 LEU A 324 34.972 −0.307 41.664 1.00 11.73 A C ATOM 292 CLEU A 324 32.519 −2.471 38.915 1.00 14.54 A C ATOM 293 O LEU A 32432.516 −1.550 38.097 1.00 13.87 A O ATOM 294 N CYS A 325 32.215 −3.72238.585 1.00 12.55 A N ATOM 295 CA CYS A 325 31.795 −4.067 37.226 1.0014.33 A C ATOM 296 CB CYS A 325 31.543 −5.568 37.097 1.00 16.72 A C ATOM297 SG CYS A 325 33.027 −6.555 37.110 1.00 14.65 A S ATOM 298 C CYS A325 30.528 −3.328 36.822 1.00 18.79 A C ATOM 299 O CYS A 325 30.468−2.717 35.749 1.00 14.81 A O ATOM 300 N ASP A 326 29.508 −3.412 37.6741.00 12.37 A N ATOM 301 CA ASP A 326 28.272 −2.685 37.437 1.00 15.44 A CATOM 302 CB ASP A 326 27.289 −2.893 38.588 1.00 19.76 A C ATOM 303 CGASP A 326 26.718 −4.296 38.619 1.00 22.47 A C ATOM 304 OD1 ASP A 32626.885 −5.044 37.628 1.00 22.18 A O ATOM 305 OD2 ASP A 326 26.098 −4.64839.642 1.00 30.32 A O ATOM 306 C ASP A 326 28.544 −1.196 37.272 1.0016.07 A C ATOM 307 O ASP A 326 28.000 −0.554 36.375 1.00 19.73 A O ATOM308 N LEU A 327 29.376 −0.641 38.148 1.00 10.96 A N ATOM 309 CA LEU A327 29.697 0.781 38.070 1.00 13.98 A C ATOM 310 CB LEU A 327 30.6171.197 39.216 1.00 10.97 A C ATOM 311 CG LEU A 327 31.149 2.627 39.1411.00 14.38 A C ATOM 312 CD1 LEU A 327 30.006 3.646 39.117 1.00 14.70 A CATOM 313 CD2 LEU A 327 32.116 2.901 40.291 1.00 10.90 A C ATOM 314 C LEUA 327 30.350 1.087 36.725 1.00 15.52 A C ATOM 315 O LEU A 327 29.9872.059 36.054 1.00 11.65 A O ATOM 316 N PHE A 328 31.296 0.232 36.3271.00 13.97 A N ATOM 317 CA PHE A 328 32.035 0.417 35.080 1.00 11.13 A CATOM 318 CB PHE A 328 33.014 −0.733 34.869 1.00 8.54 A C ATOM 319 CG PHEA 328 34.070 −0.453 33.842 1.00 13.15 A C ATOM 320 CD1 PHE A 328 34.124−1.181 32.664 1.00 12.18 A C ATOM 321 CD2 PHE A 328 35.027 0.529 34.0631.00 11.71 A C ATOM 322 CE1 PHE A 328 35.110 −0.934 31.729 1.00 15.78 AC ATOM 323 CE2 PHE A 328 36.021 0.779 33.127 1.00 11.74 A C ATOM 324 CZPHE A 328 36.062 0.053 31.960 1.00 12.05 A C ATOM 325 C PHE A 328 31.1190.534 33.870 1.00 13.77 A C ATOM 326 O PHE A 328 31.327 1.393 33.0181.00 12.07 A O ATOM 327 N ASP A 329 30.116 −0.341 33.790 1.00 12.95 A NATOM 328 CA ASP A 329 29.178 −0.326 32.671 1.00 14.28 A C ATOM 329 CBASP A 329 28.108 −1.415 32.826 1.00 14.85 A C ATOM 330 CG ASP A 32928.660 −2.817 32.617 1.00 20.42 A C ATOM 331 OD1 ASP A 329 28.069 −3.78033.149 1.00 21.88 A O ATOM 332 OD2 ASP A 329 29.685 −2.955 31.925 1.0016.14 A O ATOM 333 C ASP A 329 28.504 1.030 32.560 1.00 13.17 A C ATOM334 O ASP A 329 28.339 1.559 31.466 1.00 11.47 A O ATOM 335 N ARG A 33028.120 1.590 33.701 1.00 10.93 A N ATOM 336 CA ARG A 330 27.460 2.88733.720 1.00 13.26 A C ATOM 337 CB ARG A 330 26.799 3.122 35.080 1.0013.70 A C ATOM 338 CG ARG A 330 25.816 2.014 35.444 1.00 17.68 A C ATOM339 CD ARG A 330 24.756 2.507 36.396 1.00 27.55 A C ATOM 340 NE ARG A330 25.282 2.632 37.745 1.00 31.14 A N ATOM 341 CZ ARG A 330 24.9383.588 38.598 1.00 25.26 A C ATOM 342 NH1 ARG A 330 24.067 4.524 38.2421.00 21.90 A N ATOM 343 NH2 ARG A 330 25.477 3.609 39.806 1.00 22.06 A NATOM 344 C ARG A 330 28.423 4.023 33.361 1.00 14.84 A C ATOM 345 O ARG A330 28.032 4.983 32.690 1.00 11.00 A O ATOM 346 N GLU A 331 29.681 3.90233.792 1.00 11.85 A N ATOM 347 CA GLU A 331 30.709 4.885 33.443 1.0013.26 A C ATOM 348 CB GLU A 331 32.029 4.589 34.170 1.00 11.64 A C ATOM349 CG GLU A 331 32.103 5.126 35.592 1.00 12.18 A C ATOM 350 CD GLU A331 32.073 6.653 35.651 1.00 20.62 A C ATOM 351 OE1 GLU A 331 32.6947.311 34.774 1.00 17.66 A O ATOM 352 OE2 GLU A 331 31.433 7.191 36.5831.00 17.88 A O ATOM 353 C GLU A 331 30.965 4.914 31.945 1.00 12.27 A CATOM 354 O GLU A 331 31.266 5.956 31.385 1.00 12.04 A O ATOM 355 N ILE A332 30.857 3.763 31.294 1.00 10.67 A N ATOM 356 CA ILE A 332 31.0773.701 29.855 1.00 11.62 A C ATOM 357 CB ILE A 332 31.096 2.243 29.3531.00 13.91 A C ATOM 358 CG2 ILE A 332 30.640 2.172 27.900 1.00 10.95 A CATOM 359 CG1 ILE A 332 32.511 1.666 29.509 1.00 14.60 A C ATOM 360 CD1ILE A 332 32.546 0.170 29.645 1.00 18.15 A C ATOM 361 C ILE A 332 30.0194.497 29.100 1.00 13.24 A C ATOM 362 O ILE A 332 30.331 5.281 28.1991.00 12.76 A O ATOM 363 N VAL A 333 28.764 4.287 29.472 1.00 10.95 A NATOM 364 CA VAL A 333 27.645 4.956 28.823 1.00 11.00 A C ATOM 365 CB VALA 333 26.314 4.429 29.398 1.00 12.69 A C ATOM 366 CG1 VAL A 333 25.1445.332 28.999 1.00 12.65 A C ATOM 367 CG2 VAL A 333 26.093 2.977 28.9611.00 8.88 A C ATOM 368 C VAL A 333 27.748 6.462 29.043 1.00 13.15 A CATOM 369 O VAL A 333 27.651 7.259 28.111 1.00 15.58 A O ATOM 370 N VAL A334 27.962 6.837 30.292 1.00 9.75 A N ATOM 371 CA VAL A 334 28.130 8.22930.667 1.00 14.02 A C ATOM 372 CB VAL A 334 28.333 8.336 32.183 1.0018.06 A C ATOM 373 CG1 VAL A 334 28.975 9.645 32.540 1.00 17.29 A C ATOM374 CG2 VAL A 334 26.984 8.151 32.906 1.00 18.35 A C ATOM 375 C VAL A334 29.298 8.892 29.926 1.00 13.50 A C ATOM 376 O VAL A 334 29.20310.046 29.502 1.00 13.96 A O ATOM 377 N THR A 335 30.393 8.159 29.7601.00 9.79 A N ATOM 378 CA THR A 335 31.543 8.670 29.018 1.00 10.99 A CATOM 379 CB THR A 335 32.732 7.690 29.082 1.00 11.18 A C ATOM 380 OG1THR A 335 33.184 7.579 30.434 1.00 10.57 A O ATOM 381 CG2 THR A 33533.886 8.174 28.209 1.00 11.31 A C ATOM 382 C THR A 335 31.179 8.96227.555 1.00 11.10 A C ATOM 383 O THR A 335 31.510 10.014 27.021 1.009.77 A O ATOM 384 N ILE A 336 30.501 8.025 26.909 1.00 10.81 A N ATOM385 CA ILE A 336 30.071 8.215 25.524 1.00 14.82 A C ATOM 386 CB ILE A336 29.448 6.912 24.942 1.00 13.32 A C ATOM 387 CG2 ILE A 336 28.9037.161 23.559 1.00 15.67 A C ATOM 388 CG1 ILE A 336 30.503 5.798 24.8911.00 12.25 A C ATOM 389 CD1 ILE A 336 29.933 4.390 24.730 1.00 12.52 A CATOM 390 C ILE A 336 29.109 9.417 25.363 1.00 16.41 A C ATOM 391 O ILE A336 29.286 10.248 24.468 1.00 16.31 A O ATOM 392 N SER A 337 28.1079.518 26.234 1.00 15.70 A N ATOM 393 CA SER A 337 27.155 10.641 26.1811.00 14.63 A C ATOM 394 CB SER A 337 26.061 10.483 27.243 1.00 16.35 A CATOM 395 OG SER A 337 25.234 9.375 26.964 1.00 24.14 A O ATOM 396 C SERA 337 27.863 11.973 26.403 1.00 13.85 A C ATOM 397 O SER A 337 27.55712.985 25.755 1.00 11.00 A O ATOM 398 N TRP A 338 28.801 11.969 27.3431.00 12.03 A N ATOM 399 CA TRP A 338 29.598 13.152 27.607 1.00 12.03 A CATOM 400 CB TRP A 338 30.587 12.906 28.741 1.00 10.09 A C ATOM 401 CGTRP A 338 31.618 13.982 28.844 1.00 10.22 A C ATOM 402 CD2 TRP A 33832.943 13.960 28.287 1.00 9.82 A C ATOM 403 CE2 TRP A 338 33.558 15.17728.629 1.00 11.66 A C ATOM 404 CE3 TRP A 338 33.661 13.018 27.538 1.006.42 A C ATOM 405 CD1 TRP A 338 31.492 15.180 29.477 1.00 11.24 A C ATOM406 NE1 TRP A 338 32.654 15.904 29.357 1.00 8.68 A N ATOM 407 CZ2 TRP A338 34.864 15.487 28.241 1.00 6.65 A C ATOM 408 CZ3 TRP A 338 34.95413.324 27.157 1.00 7.40 A C ATOM 409 CH2 TRP A 338 35.544 14.551 27.5111.00 6.61 A C ATOM 410 C TRP A 338 30.335 13.602 26.347 1.00 13.94 A CATOM 411 O TRP A 338 30.237 14.766 25.954 1.00 11.68 A O ATOM 412 N ALAA 339 31.055 12.680 25.709 1.00 8.94 A N ATOM 413 CA ALA A 339 31.83213.020 24.517 1.00 10.09 A C ATOM 414 CB ALA A 339 32.538 11.796 23.9801.00 10.38 A C ATOM 415 C ALA A 339 30.996 13.667 23.413 1.00 13.70 A CATOM 416 O ALA A 339 31.460 14.594 22.737 1.00 14.16 A O ATOM 417 N LYSA 340 29.778 13.167 23.223 1.00 11.98 A N ATOM 418 CA LYS A 340 28.91213.636 22.149 1.00 16.41 A C ATOM 419 CB LYS A 340 27.699 12.722 21.9801.00 18.59 A C ATOM 420 CG LYS A 340 28.020 11.284 21.593 1.00 24.43 A CATOM 421 CD LYS A 340 26.735 10.496 21.338 1.00 29.74 A C ATOM 422 CELYS A 340 27.014 9.017 21.127 1.00 40.34 A C ATOM 423 NZ LYS A 34025.806 8.261 20.678 1.00 43.59 A N ATOM 424 C LYS A 340 28.437 15.05122.425 1.00 16.65 A C ATOM 425 O LYS A 340 28.010 15.752 21.517 1.0016.11 A O ATOM 426 N SER A 341 28.512 15.469 23.684 1.00 16.51 A N ATOM427 CA SER A 341 28.101 16.817 24.058 1.00 13.10 A C ATOM 428 CB SER A341 27.380 16.791 25.405 1.00 18.40 A C ATOM 429 OG SER A 341 28.30916.806 26.473 1.00 17.08 A O ATOM 430 C SER A 341 29.264 17.815 24.1021.00 13.99 A C ATOM 431 O SER A 341 29.082 18.974 24.473 1.00 13.97 A OATOM 432 N ILE A 342 30.459 17.370 23.733 1.00 11.84 A N ATOM 433 CA ILEA 342 31.611 18.272 23.703 1.00 11.35 A C ATOM 434 CB ILE A 342 32.94217.492 23.793 1.00 12.73 A C ATOM 435 CG2 ILE A 342 34.133 18.398 23.4811.00 8.77 A C ATOM 436 CG1 ILE A 342 33.085 16.849 25.177 1.00 10.78 A CATOM 437 CD1 ILE A 342 33.057 17.843 26.338 1.00 8.36 A C ATOM 438 C ILEA 342 31.588 19.109 22.428 1.00 12.44 A C ATOM 439 O ILE A 342 31.64418.560 21.326 1.00 12.27 A O ATOM 440 N PRO A 343 31.500 20.446 22.5691.00 10.97 A N ATOM 441 CD PRO A 343 31.567 21.224 23.817 1.00 11.85 A CATOM 442 CA PRO A 343 31.417 21.310 21.387 1.00 13.07 A C ATOM 443 CBPRO A 343 31.726 22.696 21.951 1.00 9.91 A C ATOM 444 CG PRO A 34331.279 22.636 23.361 1.00 11.17 A C ATOM 445 C PRO A 343 32.465 20.92520.350 1.00 13.44 A C ATOM 446 O PRO A 343 33.624 20.748 20.708 1.0011.20 A O ATOM 447 N GLY A 344 32.057 20.779 19.094 1.00 12.03 A N ATOM448 CA GLY A 344 32.991 20.523 18.015 1.00 15.36 A C ATOM 449 C GLY A344 33.218 19.048 17.734 1.00 15.61 A C ATOM 450 O GLY A 344 33.61318.681 16.633 1.00 19.51 A O ATOM 451 N PHE A 345 32.964 18.203 18.7281.00 14.84 A N ATOM 452 CA PHE A 345 33.097 16.762 18.553 1.00 17.20 A CATOM 453 CB PHE A 345 32.775 16.010 19.852 1.00 15.51 A C ATOM 454 CGPHE A 345 33.184 14.558 19.823 1.00 13.59 A C ATOM 455 CD1 PHE A 34534.528 14.207 19.821 1.00 10.15 A C ATOM 456 CD2 PHE A 345 32.230 13.54919.773 1.00 16.53 A C ATOM 457 CE1 PHE A 345 34.925 12.885 19.786 1.009.99 A C ATOM 458 CE2 PHE A 345 32.620 12.208 19.732 1.00 17.15 A C ATOM459 CZ PHE A 345 33.973 11.882 19.734 1.00 11.80 A C ATOM 460 C PHE A345 32.224 16.241 17.411 1.00 18.58 A C ATOM 461 O PHE A 345 32.67615.432 16.591 1.00 17.81 A O ATOM 462 N SER A 346 30.983 16.720 17.3561.00 16.25 A N ATOM 463 CA SER A 346 30.014 16.280 16.352 1.00 22.10 A CATOM 464 CB SER A 346 28.613 16.774 16.717 1.00 18.82 A C ATOM 465 OGSER A 346 28.364 16.578 18.098 1.00 37.90 A O ATOM 466 C SER A 34630.359 16.768 14.953 1.00 23.07 A C ATOM 467 O SER A 346 29.811 16.28013.965 1.00 25.31 A O ATOM 468 N SER A 347 31.248 17.748 14.869 1.0019.61 A N ATOM 469 CA SER A 347 31.689 18.245 13.574 1.00 25.27 A C ATOM470 CB SER A 347 32.466 19.553 13.728 1.00 21.25 A C ATOM 471 OG SER A347 31.626 20.577 14.238 1.00 30.98 A O ATOM 472 C SER A 347 32.54017.202 12.853 1.00 25.20 A C ATOM 473 O SER A 347 32.556 17.149 11.6221.00 25.78 A O ATOM 474 N LEU A 348 33.245 16.374 13.621 1.00 19.70 A NATOM 475 CA LEU A 348 34.041 15.293 13.045 1.00 21.27 A C ATOM 476 CBLEU A 348 34.843 14.585 14.131 1.00 16.81 A C ATOM 477 CG LEU A 34835.806 15.435 14.948 1.00 15.92 A C ATOM 478 CD1 LEU A 348 36.394 14.59616.085 1.00 13.54 A C ATOM 479 CD2 LEU A 348 36.900 16.001 14.058 1.0017.60 A C ATOM 480 C LEU A 348 33.146 14.275 12.334 1.00 19.87 A C ATOM481 O LEU A 348 31.978 14.102 12.696 1.00 16.45 A O ATOM 482 N SER A 34933.683 13.603 11.321 1.00 18.80 A N ATOM 483 CA SER A 349 32.906 12.55710.664 1.00 21.86 A C ATOM 484 CB SER A 349 33.727 11.867 9.588 1.0018.54 A C ATOM 485 OG SER A 349 34.814 11.173 10.177 1.00 20.71 A O ATOM486 C SER A 349 32.511 11.541 11.717 1.00 21.03 A C ATOM 487 O SER A 34933.231 11.345 12.701 1.00 18.54 A O ATOM 488 N LEU A 350 31.369 10.89711.519 1.00 21.90 A N ATOM 489 CA LEU A 350 30.916 9.885 12.451 1.0018.97 A C ATOM 490 CB LEU A 350 29.696 9.157 11.888 1.00 30.73 A C ATOM491 CG LEU A 350 28.662 8.614 12.878 1.00 36.96 A C ATOM 492 CD1 LEU A350 27.966 7.389 12.287 1.00 37.93 A C ATOM 493 CD2 LEU A 350 29.3008.266 14.214 1.00 34.80 A C ATOM 494 C LEU A 350 32.052 8.895 12.6871.00 21.31 A C ATOM 495 O LEU A 350 32.319 8.485 13.820 1.00 21.06 A OATOM 496 N SER A 351 32.727 8.524 11.606 1.00 22.72 A N ATOM 497 CA SERA 351 33.810 7.554 11.675 1.00 22.26 A C ATOM 498 CB SER A 351 34.3957.318 10.279 1.00 18.71 A C ATOM 499 OG SER A 351 35.400 6.319 10.3181.00 27.66 A O ATOM 500 C SER A 351 34.909 8.020 12.633 1.00 21.66 A CATOM 501 O SER A 351 35.372 7.257 13.487 1.00 22.40 A O ATOM 502 N ASP A352 35.329 9.273 12.487 1.00 20.12 A N ATOM 503 CA ASP A 352 36.3779.809 13.348 1.00 20.76 A C ATOM 504 CB ASP A 352 36.919 11.128 12.8011.00 16.76 A C ATOM 505 CG ASP A 352 37.887 10.915 11.663 1.00 21.88 A CATOM 506 OD1 ASP A 352 38.283 11.902 11.011 1.00 20.77 A O ATOM 507 OD2ASP A 352 38.255 9.744 11.428 1.00 23.51 A O ATOM 508 C ASP A 352 35.9209.957 14.794 1.00 17.01 A C ATOM 509 O ASP A 352 36.711 9.775 15.7111.00 16.19 A O ATOM 510 N GLN A 353 34.646 10.278 15.000 1.00 16.91 A NATOM 511 CA GLN A 353 34.130 10.387 16.359 1.00 15.73 A C ATOM 512 CBGLN A 353 32.641 10.740 16.362 1.00 13.27 A C ATOM 513 CG GLN A 35332.348 12.177 15.972 1.00 15.41 A C ATOM 514 CD GLN A 353 30.863 12.46215.838 1.00 21.44 A C ATOM 515 OE1 GLN A 353 30.071 12.178 16.740 1.0021.89 A O ATOM 516 NE2 GLN A 353 30.482 13.041 14.710 1.00 18.96 A NATOM 517 C GLN A 353 34.352 9.063 17.059 1.00 15.88 A C ATOM 518 O GLN A353 34.815 9.015 18.199 1.00 12.23 A O ATOM 519 N MET A 354 34.036 7.98316.355 1.00 14.37 A N ATOM 520 CA MET A 354 34.132 6.660 16.936 1.0019.04 A C ATOM 521 CB MET A 354 33.385 5.632 16.080 1.00 20.68 A C ATOM522 CG MET A 354 31.897 5.925 15.985 1.00 24.71 A C ATOM 523 SD MET A354 30.898 4.500 15.531 1.00 38.54 A S ATOM 524 CE MET A 354 31.6893.994 14.005 1.00 31.83 A C ATOM 525 C MET A 354 35.586 6.269 17.1281.00 16.32 A C ATOM 526 O MET A 354 35.942 5.699 18.160 1.00 18.95 A OATOM 527 N SER A 355 36.423 6.585 16.142 1.00 13.80 A N ATOM 528 CA SERA 355 37.845 6.277 16.223 1.00 15.36 A C ATOM 529 CB SER A 355 38.5606.625 14.915 1.00 17.70 A C ATOM 530 OG SER A 355 38.448 5.579 13.9681.00 22.38 A O ATOM 531 C SER A 355 38.501 7.024 17.381 1.00 17.93 A CATOM 532 O SER A 355 39.274 6.442 18.140 1.00 12.93 A O ATOM 533 N VAL A356 38.203 8.316 17.502 1.00 14.09 A N ATOM 534 CA VAL A 356 38.6859.100 18.634 1.00 12.55 A C ATOM 535 CB VAL A 356 38.212 10.568 18.5521.00 13.35 A C ATOM 536 CG1 VAL A 356 38.440 11.283 19.877 1.00 10.53 AC ATOM 537 CG2 VAL A 356 38.939 11.291 17.425 1.00 11.16 A C ATOM 538 CVAL A 356 38.239 8.459 19.956 1.00 14.87 A C ATOM 539 O VAL A 356 39.0698.152 20.810 1.00 10.80 A O ATOM 540 N LEU A 357 36.935 8.227 20.1101.00 10.72 A N ATOM 541 CA LEU A 357 36.415 7.661 21.351 1.00 12.32 A CATOM 542 CB LEU A 357 34.893 7.520 21.308 1.00 12.78 A C ATOM 543 CG LEUA 357 34.108 8.645 21.976 1.00 11.28 A C ATOM 544 CD1 LEU A 357 32.6218.466 21.750 1.00 18.76 A C ATOM 545 CD2 LEU A 357 34.411 8.681 23.4531.00 9.57 A C ATOM 546 C LEU A 357 37.036 6.317 21.735 1.00 13.32 A CATOM 547 O LEU A 357 37.326 6.085 22.907 1.00 11.76 A O ATOM 548 N GLN A358 37.218 5.431 20.760 1.00 11.69 A N ATOM 549 CA GLN A 358 37.7774.110 21.035 1.00 13.23 A C ATOM 550 CB GLN A 358 37.778 3.236 19.7791.00 12.80 A C ATOM 551 CG GLN A 358 36.419 2.692 19.387 1.00 18.19 A CATOM 552 CD GLN A 358 36.487 1.830 18.140 1.00 25.29 A C ATOM 553 OE1GLN A 358 36.213 2.299 17.034 1.00 28.13 A O ATOM 554 NE2 GLN A 35836.872 0.566 18.310 1.00 21.86 A N ATOM 555 C GLN A 358 39.191 4.18621.580 1.00 12.85 A C ATOM 556 O GLN A 358 39.637 3.287 22.294 1.0012.84 A O ATOM 557 N SER A 359 39.900 5.255 21.238 1.00 10.05 A N ATOM558 CA SER A 359 41.311 5.370 21.601 1.00 12.66 A C ATOM 559 CB SER A359 42.083 6.109 20.498 1.00 12.37 A C ATOM 560 OG SER A 359 41.6747.476 20.416 1.00 15.00 A O ATOM 561 C SER A 359 41.571 6.040 22.9601.00 11.14 A C ATOM 562 O SER A 359 42.627 5.833 23.558 1.00 14.86 A OATOM 563 N VAL A 360 40.622 6.837 23.445 1.00 10.02 A N ATOM 564 CA VALA 360 40.826 7.593 24.683 1.00 9.96 A C ATOM 565 CB VAL A 360 40.9619.101 24.402 1.00 9.66 A C ATOM 566 CG1 VAL A 360 42.199 9.374 23.6041.00 11.91 A C ATOM 567 CG2 VAL A 360 39.701 9.616 23.693 1.00 8.37 A CATOM 568 C VAL A 360 39.721 7.445 25.728 1.00 7.98 A C ATOM 569 O VAL A360 39.781 8.071 26.780 1.00 9.05 A O ATOM 570 N TRP A 361 38.702 6.64325.458 1.00 6.77 A N ATOM 571 CA TRP A 361 37.628 6.538 26.434 1.0011.30 A C ATOM 572 CB TRP A 361 36.540 5.553 25.989 1.00 9.72 A C ATOM573 CG TRP A 361 36.968 4.123 25.981 1.00 9.75 A C ATOM 574 CD2 TRP A361 36.840 3.183 27.055 1.00 9.74 A C ATOM 575 CE2 TRP A 361 37.3641.958 26.604 1.00 9.82 A C ATOM 576 CE3 TRP A 361 36.320 3.261 28.3531.00 11.35 A C ATOM 577 CD1 TRP A 361 37.548 3.449 24.950 1.00 12.73 A CATOM 578 NE1 TRP A 361 37.797 2.144 25.317 1.00 10.44 A N ATOM 579 CZ2TRP A 361 37.391 0.817 27.403 1.00 11.49 A C ATOM 580 CZ3 TRP A 36136.344 2.125 29.145 1.00 13.49 A C ATOM 581 CH2 TRP A 361 36.876 0.92128.666 1.00 11.95 A C ATOM 582 C TRP A 361 38.146 6.189 27.834 1.00 7.33A C ATOM 583 O TRP A 361 37.613 6.674 28.830 1.00 8.33 A O ATOM 584 NMET A 362 39.183 5.364 27.914 1.00 6.24 A N ATOM 585 CA MET A 362 39.7024.959 29.222 1.00 7.37 A C ATOM 586 CB MET A 362 40.655 3.756 29.1211.00 7.77 A C ATOM 587 CG MET A 362 41.207 3.268 30.483 1.00 9.27 A CATOM 588 SD MET A 362 39.931 2.656 31.630 1.00 10.13 A S ATOM 589 CE META 362 39.582 1.052 30.898 1.00 8.76 A C ATOM 590 C MET A 362 40.3806.136 29.915 1.00 7.87 A C ATOM 591 O MET A 362 40.265 6.291 31.131 1.008.80 A O ATOM 592 N GLU A 363 41.073 6.965 29.135 1.00 6.62 A N ATOM 593CA GLU A 363 41.684 8.189 29.656 1.00 10.64 A C ATOM 594 CB GLU A 36342.429 8.949 28.549 1.00 6.65 A C ATOM 595 CG GLU A 363 43.655 8.23228.003 1.00 9.31 A C ATOM 596 CD GLU A 363 44.474 9.106 27.056 1.0014.27 A C ATOM 597 OE1 GLU A 363 45.155 8.533 26.175 1.00 13.30 A O ATOM598 OE2 GLU A 363 44.439 10.357 27.186 1.00 10.35 A O ATOM 599 C GLU A363 40.630 9.106 30.281 1.00 6.33 A C ATOM 600 O GLU A 363 40.801 9.62331.389 1.00 5.31 A O ATOM 601 N VAL A 364 39.548 9.332 29.551 1.00 5.93A N ATOM 602 CA VAL A 364 38.477 10.177 30.064 1.00 5.96 A C ATOM 603 CBVAL A 364 37.344 10.308 29.028 1.00 6.40 A C ATOM 604 CG1 VAL A 36436.125 10.998 29.644 1.00 6.59 A C ATOM 605 CG2 VAL A 364 37.847 11.05827.775 1.00 7.12 A C ATOM 606 C VAL A 364 37.941 9.589 31.381 1.00 7.64A C ATOM 607 O VAL A 364 37.817 10.284 32.400 1.00 7.59 A O ATOM 608 NLEU A 365 37.636 8.300 31.351 1.00 6.22 A N ATOM 609 CA LEU A 365 37.0757.609 32.506 1.00 7.44 A C ATOM 610 CB LEU A 365 36.830 6.139 32.1501.00 8.63 A C ATOM 611 CG LEU A 365 35.820 5.378 33.005 1.00 9.53 A CATOM 612 CD1 LEU A 365 35.258 4.207 32.225 1.00 9.09 A C ATOM 613 CD2LEU A 365 36.462 4.913 34.308 1.00 10.31 A C ATOM 614 C LEU A 365 38.0177.710 33.702 1.00 8.08 A C ATOM 615 O LEU A 365 37.618 8.119 34.797 1.005.69 A O ATOM 616 N VAL A 366 39.276 7.342 33.482 1.00 8.82 A N ATOM 617CA VAL A 366 40.279 7.382 34.538 1.00 6.69 A C ATOM 618 CB VAL A 36641.636 6.800 34.055 1.00 9.23 A C ATOM 619 CG1 VAL A 366 42.792 7.36734.875 1.00 6.28 A C ATOM 620 CG2 VAL A 366 41.600 5.285 34.145 1.008.23 A C ATOM 621 C VAL A 366 40.467 8.791 35.099 1.00 6.44 A C ATOM 622O VAL A 366 40.662 8.976 36.307 1.00 6.79 A O ATOM 623 N LEU A 36740.409 9.795 34.234 1.00 6.54 A N ATOM 624 CA LEU A 367 40.563 11.16734.720 1.00 8.51 A C ATOM 625 CB LEU A 367 40.660 12.164 33.558 1.005.56 A C ATOM 626 CG LEU A 367 40.932 13.612 33.982 1.00 8.69 A C ATOM627 CD1 LEU A 367 42.249 13.685 34.727 1.00 3.58 A C ATOM 628 CD2 LEU A367 40.947 14.545 32.763 1.00 7.76 A C ATOM 629 C LEU A 367 39.40511.527 35.667 1.00 6.52 A C ATOM 630 O LEU A 367 39.588 12.243 36.6431.00 7.03 A O ATOM 631 N GLY A 368 38.215 11.026 35.375 1.00 4.77 A NATOM 632 CA GLY A 368 37.083 11.211 36.270 1.00 10.05 A C ATOM 633 C GLYA 368 37.357 10.643 37.654 1.00 8.34 A C ATOM 634 O GLY A 368 37.16111.322 38.667 1.00 9.15 A O ATOM 635 N VAL A 369 37.816 9.395 37.6971.00 8.95 A N ATOM 636 CA VAL A 369 38.204 8.743 38.959 1.00 9.48 A CATOM 637 CB VAL A 369 38.794 7.345 38.713 1.00 5.72 A C ATOM 638 CG1 VALA 369 39.399 6.780 40.010 1.00 5.85 A C ATOM 639 CG2 VAL A 369 37.7286.385 38.121 1.00 8.99 A C ATOM 640 C VAL A 369 39.237 9.564 39.744 1.007.51 A C ATOM 641 O VAL A 369 39.108 9.766 40.953 1.00 6.01 A O ATOM 642N ALA A 370 40.268 10.026 39.049 1.00 7.55 A N ATOM 643 CA ALA A 37041.318 10.818 39.689 1.00 6.33 A C ATOM 644 CB ALA A 370 42.427 11.11038.699 1.00 5.48 A C ATOM 645 C ALA A 370 40.704 12.118 40.210 1.00 9.15A C ATOM 646 O ALA A 370 40.992 12.568 41.321 1.00 6.32 A O ATOM 647 NGLN A 371 39.830 12.695 39.394 1.00 8.46 A N ATOM 648 CA GLN A 37139.153 13.936 39.721 1.00 9.76 A C ATOM 649 CB GLN A 371 38.249 14.34238.544 1.00 11.97 A C ATOM 650 CG GLN A 371 36.838 14.762 38.941 1.0021.47 A C ATOM 651 CD GLN A 371 36.865 16.064 39.645 1.00 20.86 A C ATOM652 OE1 GLN A 371 37.842 16.799 39.527 1.00 30.40 A O ATOM 653 NE2 GLN A371 35.812 16.372 40.394 1.00 24.96 A N ATOM 654 C GLN A 371 38.36113.805 41.027 1.00 10.44 A C ATOM 655 O GLN A 371 38.427 14.670 41.8971.00 10.03 A O ATOM 656 N ARG A 372 37.611 12.718 41.173 1.00 9.65 A NATOM 657 CA ARG A 372 36.831 12.524 42.397 1.00 11.43 A C ATOM 658 CBARG A 372 35.792 11.410 42.217 1.00 10.17 A C ATOM 659 CG ARG A 37234.853 11.627 41.045 1.00 7.35 A C ATOM 660 CD ARG A 372 33.726 10.59041.008 1.00 10.71 A C ATOM 661 NE ARG A 372 34.136 9.296 40.466 1.009.30 A N ATOM 662 CZ ARG A 372 34.323 9.048 39.169 1.00 14.09 A C ATOM663 NH1 ARG A 372 34.162 10.017 38.266 1.00 9.78 A N ATOM 664 NH2 ARG A372 34.678 7.828 38.766 1.00 11.35 A N ATOM 665 C ARG A 372 37.72612.229 43.610 1.00 11.57 A C ATOM 666 O ARG A 372 37.304 12.384 44.7531.00 10.98 A O ATOM 667 N SER A 373 38.965 11.818 43.357 1.00 6.73 A NATOM 668 CA SER A 373 39.870 11.431 44.433 1.00 8.65 A C ATOM 669 CB SERA 373 40.807 10.302 43.969 1.00 10.24 A C ATOM 670 OG SER A 373 40.0739.218 43.415 1.00 9.92 A O ATOM 671 C SER A 373 40.697 12.597 44.9731.00 9.36 A C ATOM 672 O SER A 373 41.357 12.465 46.010 1.00 8.35 A OATOM 673 N LEU A 374 40.663 13.738 44.283 1.00 9.32 A N ATOM 674 CA LEUA 374 41.542 14.853 44.649 1.00 10.69 A C ATOM 675 CB LEU A 374 41.40216.046 43.684 1.00 10.73 A C ATOM 676 CG LEU A 374 41.741 15.856 42.1951.00 10.64 A C ATOM 677 CD1 LEU A 374 41.658 17.182 41.464 1.00 9.35 A CATOM 678 CD2 LEU A 374 43.107 15.230 41.985 1.00 9.59 A C ATOM 679 C LEUA 374 41.424 15.321 46.110 1.00 8.24 A C ATOM 680 O LEU A 374 42.43315.642 46.721 1.00 9.80 A O ATOM 681 N PRO A 375 40.203 15.379 46.6681.00 8.86 A N ATOM 682 CD PRO A 375 38.887 15.308 46.014 1.00 14.08 A CATOM 683 CA PRO A 375 40.105 15.902 48.043 1.00 13.75 A C ATOM 684 CBPRO A 375 38.609 16.215 48.207 1.00 14.41 A C ATOM 685 CG PRO A 37538.065 16.291 46.810 1.00 17.04 A C ATOM 686 C PRO A 375 40.542 14.89849.106 1.00 14.57 A C ATOM 687 O PRO A 375 40.662 15.248 50.286 1.0011.98 A O ATOM 688 N LEU A 376 40.797 13.666 48.687 1.00 11.24 A N ATOM689 CA LEU A 376 41.072 12.597 49.627 1.00 10.05 A C ATOM 690 CB LEU A376 40.369 11.310 49.175 1.00 11.85 A C ATOM 691 CG LEU A 376 38.85511.445 48.930 1.00 13.18 A C ATOM 692 CD1 LEU A 376 38.250 10.148 48.3771.00 11.14 A C ATOM 693 CD2 LEU A 376 38.122 11.868 50.205 1.00 9.73 A CATOM 694 C LEU A 376 42.578 12.397 49.740 1.00 14.10 A C ATOM 695 O LEUA 376 43.341 12.984 48.982 1.00 12.26 A O ATOM 696 N GLN A 377 43.00711.594 50.706 1.00 14.87 A N ATOM 697 CA GLN A 377 44.416 11.276 50.8331.00 16.83 A C ATOM 698 CB GLN A 377 44.982 11.782 52.160 1.00 24.46 A CATOM 699 CG GLN A 377 46.275 11.063 52.563 1.00 33.92 A C ATOM 700 CDGLN A 377 47.085 11.817 53.618 1.00 54.60 A C ATOM 701 OE1 GLN A 37747.531 11.237 54.614 1.00 48.37 A O ATOM 702 NE2 GLN A 377 47.281 13.11653.397 1.00 60.36 A N ATOM 703 C GLN A 377 44.637 9.776 50.700 1.0015.47 A C ATOM 704 O GLN A 377 44.196 8.996 51.539 1.00 15.74 A O ATOM705 N ASP A 378 45.316 9.384 49.628 1.00 15.96 A N ATOM 706 CA ASP A 37845.640 7.982 49.382 1.00 19.65 A C ATOM 707 CB ASP A 378 46.539 7.44350.485 1.00 14.84 A C ATOM 708 CG ASP A 378 47.819 8.233 50.614 1.0020.81 A C ATOM 709 OD1 ASP A 378 48.295 8.436 51.751 1.00 28.90 A O ATOM710 OD2 ASP A 378 48.344 8.669 49.574 1.00 22.14 A O ATOM 711 C ASP A378 44.386 7.132 49.262 1.00 14.53 A C ATOM 712 O ASP A 378 44.368 5.97449.665 1.00 16.70 A O ATOM 713 N GLU A 379 43.340 7.724 48.705 1.0013.21 A N ATOM 714 CA GLU A 379 42.102 7.009 48.482 1.00 12.77 A C ATOM715 CB GLU A 379 41.055 7.413 49.514 1.00 12.81 A C ATOM 716 CG GLU A379 41.411 7.010 50.937 1.00 20.07 A C ATOM 717 CD GLU A 379 40.2717.258 51.907 1.00 20.55 A C ATOM 718 OE1 GLU A 379 39.800 8.412 52.0011.00 19.77 A O ATOM 719 OE2 GLU A 379 39.841 6.292 52.570 1.00 30.93 A OATOM 720 C GLU A 379 41.587 7.303 47.092 1.00 13.51 A C ATOM 721 O GLU A379 41.897 8.344 46.510 1.00 12.64 A O ATOM 722 N LEU A 380 40.786 6.37946.578 1.00 14.16 A N ATOM 723 CA LEU A 380 40.207 6.486 45.251 1.0011.49 A C ATOM 724 CB LEU A 380 40.681 5.314 44.388 1.00 13.39 A C ATOM725 CG LEU A 380 42.194 5.232 44.123 1.00 8.17 A C ATOM 726 CD1 LEU A380 42.589 3.869 43.547 1.00 9.15 A C ATOM 727 CD2 LEU A 380 42.6476.373 43.205 1.00 6.52 A C ATOM 728 C LEU A 380 38.682 6.488 45.367 1.0014.12 A C ATOM 729 O LEU A 380 38.086 5.537 45.882 1.00 10.80 A O ATOM730 N ALA A 381 38.062 7.575 44.910 1.00 11.42 A N ATOM 731 CA ALA A 38136.608 7.705 44.926 1.00 11.52 A C ATOM 732 CB ALA A 381 36.194 9.15545.184 1.00 6.66 A C ATOM 733 C ALA A 381 36.053 7.231 43.601 1.00 9.79A C ATOM 734 O ALA A 381 35.817 8.023 42.682 1.00 9.06 A O ATOM 735 NPHE A 382 35.869 5.928 43.492 1.00 10.68 A N ATOM 736 CA PHE A 38235.320 5.356 42.276 1.00 12.09 A C ATOM 737 CB PHE A 382 35.382 3.82642.332 1.00 10.44 A C ATOM 738 CG PHE A 382 36.769 3.279 42.138 1.0011.39 A C ATOM 739 CD1 PHE A 382 37.560 2.945 43.230 1.00 10.96 A C ATOM740 CD2 PHE A 382 37.295 3.133 40.867 1.00 10.93 A C ATOM 741 CE1 PHE A382 38.843 2.444 43.054 1.00 13.01 A C ATOM 742 CE2 PHE A 382 38.5842.637 40.677 1.00 12.78 A C ATOM 743 CZ PHE A 382 39.360 2.293 41.7711.00 12.04 A C ATOM 744 C PHE A 382 33.907 5.870 42.048 1.00 11.39 A CATOM 745 O PHE A 382 33.460 5.984 40.912 1.00 10.81 A O ATOM 746 N ALA A383 33.223 6.200 43.144 1.00 10.40 A N ATOM 747 CA ALA A 383 31.8936.791 43.090 1.00 17.44 A C ATOM 748 CB ALA A 383 30.828 5.728 42.8371.00 16.71 A C ATOM 749 C ALA A 383 31.629 7.510 44.401 1.00 17.54 A CATOM 750 O ALA A 383 32.380 7.349 45.362 1.00 15.38 A O ATOM 751 N GLU A384 30.561 8.302 44.433 1.00 23.55 A N ATOM 752 CA GLU A 384 30.1949.043 45.637 1.00 30.68 A C ATOM 753 CB GLU A 384 28.766 9.588 45.5071.00 36.59 A C ATOM 754 CG GLU A 384 28.405 10.665 46.521 1.00 41.29 A CATOM 755 CD GLU A 384 29.191 11.950 46.313 1.00 51.97 A C ATOM 756 OE1GLU A 384 29.494 12.288 45.145 1.00 51.70 A O ATOM 757 OE2 GLU A 38429.505 12.622 47.319 1.00 45.08 A O ATOM 758 C GLU A 384 30.310 8.14746.872 1.00 27.66 A C ATOM 759 O GLU A 384 30.874 8.542 47.891 1.0030.68 A O ATOM 760 N ASP A 385 29.808 6.924 46.748 1.00 24.97 A N ATOM761 CA ASP A 385 29.681 6.007 47.873 1.00 22.29 A C ATOM 762 CB ASP A385 28.237 5.513 47.949 1.00 28.50 A C ATOM 763 CG ASP A 385 27.7824.867 46.654 1.00 28.92 A C ATOM 764 OD1 ASP A 385 26.674 4.293 46.6251.00 31.32 A O ATOM 765 OD2 ASP A 385 28.540 4.938 45.658 1.00 23.99 A OATOM 766 C ASP A 385 30.595 4.794 47.748 1.00 25.57 A C ATOM 767 O ASP A385 30.310 3.739 48.311 1.00 26.40 A O ATOM 768 N LEU A 386 31.686 4.92747.005 1.00 21.78 A N ATOM 769 CA LEU A 386 32.617 3.820 46.859 1.0019.10 A C ATOM 770 CB LEU A 386 32.269 2.984 45.624 1.00 17.20 A C ATOM771 CG LEU A 386 33.230 1.865 45.204 1.00 24.05 A C ATOM 772 CD1 LEU A386 33.784 1.113 46.396 1.00 21.63 A C ATOM 773 CD2 LEU A 386 32.5600.908 44.215 1.00 20.24 A C ATOM 774 C LEU A 386 34.045 4.351 46.8101.00 18.71 A C ATOM 775 O LEU A 386 34.524 4.809 45.772 1.00 12.82 A OATOM 776 N VAL A 387 34.703 4.299 47.960 1.00 17.48 A N ATOM 777 CA VALA 387 36.012 4.904 48.153 1.00 15.69 A C ATOM 778 CB VAL A 387 35.9446.009 49.215 1.00 16.36 A C ATOM 779 CG1 VAL A 387 37.351 6.457 49.6051.00 16.49 A C ATOM 780 CG2 VAL A 387 35.077 7.178 48.734 1.00 16.54 A CATOM 781 C VAL A 387 36.943 3.839 48.683 1.00 15.82 A C ATOM 782 O VAL A387 36.677 3.260 49.735 1.00 19.22 A O ATOM 783 N LEU A 388 38.029 3.57747.963 1.00 15.55 A N ATOM 784 CA LEU A 388 38.949 2.501 48.318 1.0015.32 A C ATOM 785 CB LEU A 388 38.951 1.439 47.222 1.00 13.11 A C ATOM786 CG LEU A 388 37.621 0.784 46.870 1.00 14.26 A C ATOM 787 CD1 LEU A388 37.802 −0.144 45.667 1.00 14.40 A C ATOM 788 CD2 LEU A 388 37.0740.023 48.078 1.00 15.81 A C ATOM 789 C LEU A 388 40.371 3.016 48.4791.00 17.45 A C ATOM 790 O LEU A 388 40.804 3.883 47.722 1.00 15.60 A OATOM 791 N ASP A 389 41.095 2.485 49.462 1.00 14.27 A N ATOM 792 CA ASPA 389 42.524 2.740 49.552 1.00 15.01 A C ATOM 793 CB ASP A 389 43.0232.770 51.007 1.00 15.97 A C ATOM 794 CG ASP A 389 42.771 1.461 51.7621.00 22.33 A C ATOM 795 OD1 ASP A 389 43.057 1.432 52.979 1.00 23.29 A OATOM 796 OD2 ASP A 389 42.291 0.469 51.165 1.00 19.43 A O ATOM 797 C ASPA 389 43.231 1.676 48.730 1.00 14.30 A C ATOM 798 O ASP A 389 42.5790.846 48.097 1.00 16.58 A O ATOM 799 N GLU A 390 44.553 1.698 48.7261.00 15.09 A N ATOM 800 CA GLU A 390 45.310 0.770 47.895 1.00 16.00 A CATOM 801 CB GLU A 390 46.806 0.987 48.094 1.00 18.13 A C ATOM 802 CG GLUA 390 47.643 0.358 47.027 1.00 22.46 A C ATOM 803 CD GLU A 390 49.0770.826 47.065 1.00 27.40 A C ATOM 804 OE1 GLU A 390 49.333 1.960 47.5431.00 28.62 A O ATOM 805 OE2 GLU A 390 49.944 0.055 46.604 1.00 27.35 A OATOM 806 C GLU A 390 44.941 −0.688 48.183 1.00 21.66 A C ATOM 807 O GLUA 390 44.725 −1.478 47.260 1.00 20.71 A O ATOM 808 N GLU A 391 44.861−1.037 49.464 1.00 19.04 A N ATOM 809 CA GLU A 391 44.582 −2.411 49.8591.00 24.03 A C ATOM 810 CB GLU A 391 44.797 −2.592 51.359 1.00 27.75 A CATOM 811 CG GLU A 391 46.261 −2.509 51.758 1.00 38.64 A C ATOM 812 CDGLU A 391 46.483 −2.757 53.241 1.00 57.74 A C ATOM 813 OE1 GLU A 39145.488 −2.768 54.003 1.00 48.08 A O ATOM 814 OE2 GLU A 391 47.659 −2.94053.638 1.00 53.93 A O ATOM 815 C GLU A 391 43.177 −2.833 49.481 1.0020.44 A C ATOM 816 O GLU A 391 42.947 −3.981 49.100 1.00 19.46 A O ATOM817 N GLY A 392 42.240 −1.900 49.592 1.00 15.59 A N ATOM 818 CA GLY A392 40.868 −2.148 49.198 1.00 18.72 A C ATOM 819 C GLY A 392 40.736−2.367 47.699 1.00 16.46 A C ATOM 820 O GLY A 392 39.908 −3.167 47.2571.00 13.18 A O ATOM 821 N ALA A 393 41.542 −1.656 46.912 1.00 13.40 A NATOM 822 CA ALA A 393 41.533 −1.835 45.461 1.00 12.07 A C ATOM 823 CBALA A 393 42.354 −0.746 44.771 1.00 12.61 A C ATOM 824 C ALA A 39342.075 −3.215 45.109 1.00 14.92 A C ATOM 825 O ALA A 393 41.496 −3.93844.297 1.00 12.11 A O ATOM 826 N ARG A 394 43.189 −3.568 45.735 1.0011.42 A N ATOM 827 CA ARG A 394 43.808 −4.863 45.531 1.00 15.97 A C ATOM828 CB ARG A 394 45.048 −4.967 46.421 1.00 17.24 A C ATOM 829 CG ARG A394 46.048 −6.024 46.019 1.00 25.58 A C ATOM 830 CD ARG A 394 47.479−5.481 46.137 1.00 29.11 A C ATOM 831 NE ARG A 394 47.679 −4.667 47.3361.00 27.07 A N ATOM 832 CZ ARG A 394 48.580 −3.691 47.427 1.00 33.82 A CATOM 833 NH1 ARG A 394 49.349 −3.399 46.385 1.00 34.86 A N ATOM 834 NH2ARG A 394 48.706 −2.995 48.553 1.00 29.51 A N ATOM 835 C ARG A 39442.798 −5.969 45.848 1.00 16.73 A C ATOM 836 O ARG A 394 42.657 −6.93345.099 1.00 18.65 A O ATOM 837 N ALA A 395 42.074 −5.807 46.947 1.0014.18 A N ATOM 838 CA ALA A 395 41.106 −6.812 47.372 1.00 16.13 A C ATOM839 CB ALA A 395 40.716 −6.597 48.845 1.00 12.32 A C ATOM 840 C ALA A395 39.871 −6.799 46.480 1.00 19.06 A C ATOM 841 O ALA A 395 39.083−7.750 46.481 1.00 16.59 A O ATOM 842 N ALA A 396 39.702 −5.725 45.7101.00 14.54 A N ATOM 843 CA ALA A 396 38.552 −5.624 44.812 1.00 12.53 A CATOM 844 CB ALA A 396 37.994 −4.197 44.802 1.00 15.65 A C ATOM 845 C ALAA 396 38.891 −6.081 43.394 1.00 13.40 A C ATOM 846 O ALA A 396 38.122−5.859 42.465 1.00 18.31 A O ATOM 847 N GLY A 397 40.049 −6.708 43.2351.00 14.85 A N ATOM 848 CA GLY A 397 40.434 −7.296 41.965 1.00 14.90 A CATOM 849 C GLY A 397 41.245 −6.398 41.037 1.00 17.60 A C ATOM 850 O GLYA 397 41.512 −6.772 39.897 1.00 16.08 A O ATOM 851 N LEU A 398 41.654−5.229 41.527 1.00 15.81 A N ATOM 852 CA LEU A 398 42.314 −4.220 40.6901.00 14.58 A C ATOM 853 CB LEU A 398 41.949 −2.815 41.184 1.00 10.85 A CATOM 854 CG LEU A 398 40.461 −2.478 41.075 1.00 9.27 A C ATOM 855 CD1LEU A 398 40.122 −1.134 41.713 1.00 10.67 A C ATOM 856 CD2 LEU A 39840.066 −2.479 39.620 1.00 14.03 A C ATOM 857 C LEU A 398 43.837 −4.37740.629 1.00 16.08 A C ATOM 858 O LEU A 398 44.545 −3.537 40.046 1.0014.37 A O ATOM 859 N GLY A 399 44.339 −5.445 41.234 1.00 12.30 A N ATOM860 CA GLY A 399 45.766 −5.699 41.253 1.00 11.79 A C ATOM 861 C GLY A399 46.505 −4.499 41.804 1.00 14.14 A C ATOM 862 O GLY A 399 46.197−4.021 42.899 1.00 15.51 A O ATOM 863 N GLU A 400 47.465 −3.996 41.0371.00 12.60 A N ATOM 864 CA GLU A 400 48.245 −2.836 41.455 1.00 13.99 A CATOM 865 CB GLU A 400 49.720 −3.026 41.096 1.00 17.29 A C ATOM 866 CGGLU A 400 50.402 −4.170 41.847 1.00 23.15 A C ATOM 867 CD GLU A 40050.218 −4.061 43.350 1.00 25.72 A C ATOM 868 OE1 GLU A 400 49.736 −5.03843.959 1.00 31.69 A O ATOM 869 OE2 GLU A 400 50.535 −2.996 43.921 1.0030.47 A O ATOM 870 C GLU A 400 47.726 −1.537 40.842 1.00 12.72 A C ATOM871 O GLU A 400 48.413 −0.514 40.876 1.00 11.57 A O ATOM 872 N LEU A 40146.525 −1.574 40.269 1.00 11.68 A N ATOM 873 CA LEU A 401 45.950 −0.37339.679 1.00 11.44 A C ATOM 874 CB LEU A 401 44.642 −0.668 38.940 1.0010.03 A C ATOM 875 CG LEU A 401 44.742 −1.526 37.678 1.00 10.87 A C ATOM876 CD1 LEU A 401 43.359 −1.748 37.072 1.00 10.54 A C ATOM 877 CD2 LEU A401 45.694 −0.902 36.671 1.00 10.80 A C ATOM 878 C LEU A 401 45.7250.672 40.767 1.00 14.90 A C ATOM 879 O LEU A 401 45.772 1.877 40.5041.00 12.23 A O ATOM 880 N GLY A 402 45.478 0.211 41.989 1.00 11.10 A NATOM 881 CA GLY A 402 45.332 1.123 43.105 1.00 8.33 A C ATOM 882 C GLY A402 46.511 2.077 43.160 1.00 12.05 A C ATOM 883 O GLY A 402 46.341 3.29643.220 1.00 9.18 A O ATOM 884 N ALA A 403 47.715 1.520 43.103 1.00 10.25A N ATOM 885 CA ALA A 403 48.929 2.322 43.156 1.00 12.95 A C ATOM 886 CBALA A 403 50.162 1.424 43.288 1.00 11.24 A C ATOM 887 C ALA A 403 49.0553.223 41.926 1.00 10.76 A C ATOM 888 O ALA A 403 49.405 4.394 42.0431.00 9.68 A O ATOM 889 N ALA A 404 48.764 2.677 40.750 1.00 8.99 A NATOM 890 CA ALA A 404 48.915 3.427 39.501 1.00 10.80 A C ATOM 891 CB ALAA 404 48.695 2.509 38.303 1.00 10.92 A C ATOM 892 C ALA A 404 47.9544.621 39.442 1.00 12.45 A C ATOM 893 O ALA A 404 48.324 5.740 39.0521.00 9.31 A O ATOM 894 N LEU A 405 46.709 4.373 39.821 1.00 11.85 A NATOM 895 CA LEU A 405 45.714 5.431 39.853 1.00 8.39 A C ATOM 896 CB LEUA 405 44.340 4.853 40.168 1.00 8.93 A C ATOM 897 CG LEU A 405 43.7354.076 39.002 1.00 10.00 A C ATOM 898 CD1 LEU A 405 42.556 3.245 39.4671.00 7.23 A C ATOM 899 CD2 LEU A 405 43.321 5.047 37.903 1.00 9.72 A CATOM 900 C LEU A 405 46.097 6.515 40.860 1.00 9.64 A C ATOM 901 O LEU A405 46.031 7.704 40.545 1.00 12.51 A O ATOM 902 N LEU A 406 46.512 6.11542.058 1.00 9.03 A N ATOM 903 CA LEU A 406 46.928 7.092 43.072 1.0012.76 A C ATOM 904 CB LEU A 406 47.231 6.417 44.415 1.00 11.94 A C ATOM905 CG LEU A 406 46.008 5.984 45.225 1.00 13.22 A C ATOM 906 CD1 LEU A406 46.413 5.086 46.393 1.00 11.84 A C ATOM 907 CD2 LEU A 406 45.2367.212 45.705 1.00 9.96 A C ATOM 908 C LEU A 406 48.131 7.912 42.611 1.009.76 A C ATOM 909 O LEU A 406 48.287 9.070 42.989 1.00 11.41 A O ATOM910 N GLN A 407 48.986 7.308 41.796 1.00 10.47 A N ATOM 911 CA GLN A 40750.108 8.037 41.220 1.00 10.23 A C ATOM 912 CB GLN A 407 50.944 7.10540.357 1.00 12.87 A C ATOM 913 CG GLN A 407 52.308 7.650 40.016 1.0018.46 A C ATOM 914 CD GLN A 407 53.175 6.612 39.326 1.00 27.96 A C ATOM915 OE1 GLN A 407 52.811 5.433 39.240 1.00 26.97 A O ATOM 916 NE2 GLN A407 54.323 7.047 38.825 1.00 23.36 A N ATOM 917 C GLN A 407 49.596 9.20440.373 1.00 10.26 A C ATOM 918 O GLN A 407 50.103 10.325 40.467 1.009.77 A O ATOM 919 N LEU A 408 48.584 8.931 39.551 1.00 9.23 A N ATOM 920CA LEU A 408 47.954 9.964 38.729 1.00 8.47 A C ATOM 921 CB LEU A 40846.860 9.351 37.839 1.00 7.74 A C ATOM 922 CG LEU A 408 46.445 10.00036.513 1.00 8.05 A C ATOM 923 CD1 LEU A 408 44.963 9.764 36.197 1.004.98 A C ATOM 924 CD2 LEU A 408 46.787 11.473 36.412 1.00 6.46 A C ATOM925 C LEU A 408 47.329 11.021 39.636 1.00 8.66 A C ATOM 926 O LEU A 40847.501 12.218 39.414 1.00 9.51 A O ATOM 927 N VAL A 409 46.588 10.56840.647 1.00 7.46 A N ATOM 928 CA VAL A 409 45.906 11.465 41.579 1.006.92 A C ATOM 929 CB VAL A 409 45.157 10.668 42.686 1.00 8.65 A C ATOM930 CG1 VAL A 409 44.547 11.603 43.711 1.00 8.71 A C ATOM 931 CG2 VAL A409 44.079 9.777 42.078 1.00 8.89 A C ATOM 932 C VAL A 409 46.880 12.44842.227 1.00 11.22 A C ATOM 933 O VAL A 409 46.592 13.641 42.317 1.0011.95 A O ATOM 934 N ARG A 410 48.031 11.941 42.672 1.00 8.64 A N ATOM935 CA ARG A 410 49.041 12.766 43.329 1.00 11.83 A C ATOM 936 CB ARG A410 50.168 11.894 43.909 1.00 11.23 A C ATOM 937 CG ARG A 410 49.74311.103 45.159 1.00 15.02 A C ATOM 938 CD ARG A 410 50.939 10.572 45.9321.00 13.71 A C ATOM 939 NE ARG A 410 51.751 9.649 45.136 1.00 16.06 A NATOM 940 CZ ARG A 410 51.467 8.359 44.959 1.00 16.13 A C ATOM 941 NH1ARG A 410 50.384 7.830 45.513 1.00 13.28 A N ATOM 942 NH2 ARG A 41052.265 7.594 44.224 1.00 12.54 A N ATOM 943 C ARG A 410 49.615 13.80442.378 1.00 11.91 A C ATOM 944 O ARG A 410 49.854 14.949 42.756 1.0013.27 A O ATOM 945 N ARG A 411 49.818 13.402 41.129 1.00 12.33 A N ATOM946 CA ARG A 411 50.325 14.314 40.103 1.00 12.23 A C ATOM 947 CB ARG A411 50.560 13.530 38.808 1.00 13.09 A C ATOM 948 CG ARG A 411 51.19314.305 37.701 1.00 14.85 A C ATOM 949 CD ARG A 411 52.703 14.473 37.8771.00 10.64 A C ATOM 950 NE ARG A 411 53.121 15.589 37.036 1.00 9.23 A NATOM 951 CZ ARG A 411 53.941 15.504 36.000 1.00 8.72 A C ATOM 952 NH1ARG A 411 54.511 14.341 35.669 1.00 6.36 A N ATOM 953 NH2 ARG A 41154.207 16.604 35.314 1.00 5.92 A N ATOM 954 C ARG A 411 49.362 15.49739.890 1.00 11.17 A C ATOM 955 O ARG A 411 49.786 16.637 39.668 1.0011.92 A O ATOM 956 N LEU A 412 48.065 15.234 39.990 1.00 9.22 A N ATOM957 CA LEU A 412 47.065 16.289 39.841 1.00 12.80 A C ATOM 958 CB LEU A412 45.737 15.707 39.365 1.00 8.02 A C ATOM 959 CG LEU A 412 45.75915.057 37.988 1.00 10.32 A C ATOM 960 CD1 LEU A 412 44.492 14.244 37.7741.00 6.91 A C ATOM 961 CD2 LEU A 412 45.905 16.118 36.906 1.00 10.21 A CATOM 962 C LEU A 412 46.856 17.101 41.121 1.00 11.20 A C ATOM 963 O LEUA 412 46.626 18.310 41.066 1.00 14.93 A O ATOM 964 N GLN A 413 46.92416.435 42.265 1.00 11.94 A N ATOM 965 CA GLN A 413 46.783 17.107 43.5531.00 13.74 A C ATOM 966 CB GLN A 413 46.871 16.109 44.711 1.00 10.70 A CATOM 967 CG GLN A 413 45.653 15.226 44.897 1.00 13.99 A C ATOM 968 CDGLN A 413 45.853 14.228 46.023 1.00 14.92 A C ATOM 969 OE1 GLN A 41346.919 13.621 46.142 1.00 13.57 A O ATOM 970 NE2 GLN A 413 44.836 14.06546.863 1.00 11.26 A N ATOM 971 C GLN A 413 47.878 18.145 43.722 1.0012.17 A C ATOM 972 O GLN A 413 47.668 19.178 44.349 1.00 15.85 A O ATOM973 N ALA A 414 49.051 17.867 43.163 1.00 11.15 A N ATOM 974 CA ALA A414 50.180 18.793 43.255 1.00 11.85 A C ATOM 975 CB ALA A 414 51.42818.168 42.634 1.00 12.32 A C ATOM 976 C ALA A 414 49.886 20.149 42.5961.00 12.81 A C ATOM 977 O ALA A 414 50.496 21.152 42.944 1.00 13.14 A OATOM 978 N LEU A 415 48.962 20.175 41.641 1.00 14.00 A N ATOM 979 CA LEUA 415 48.648 21.409 40.922 1.00 12.59 A C ATOM 980 CB LEU A 415 48.37521.118 39.443 1.00 12.24 A C ATOM 981 CG LEU A 415 49.452 20.343 38.6781.00 15.07 A C ATOM 982 CD1 LEU A 415 49.027 20.137 37.238 1.00 13.21 AC ATOM 983 CD2 LEU A 415 50.800 21.055 38.760 1.00 11.85 A C ATOM 984 CLEU A 415 47.444 22.126 41.536 1.00 16.77 A C ATOM 985 O LEU A 41547.148 23.279 41.191 1.00 10.65 A O ATOM 986 N ARG A 416 46.747 21.43442.435 1.00 13.53 A N ATOM 987 CA ARG A 416 45.525 21.975 43.034 1.0017.12 A C ATOM 988 CB ARG A 416 45.879 23.090 44.026 1.00 18.84 A C ATOM989 CG ARG A 416 46.696 22.579 45.215 1.00 25.04 A C ATOM 990 CD ARG A416 47.264 23.701 46.102 1.00 42.45 A C ATOM 991 NE ARG A 416 47.93623.161 47.290 1.00 55.20 A N ATOM 992 CZ ARG A 416 48.633 23.882 48.1701.00 58.48 A C ATOM 993 NH1 ARG A 416 48.764 25.195 48.013 1.00 57.23 AN ATOM 994 NH2 ARG A 416 49.204 23.289 49.215 1.00 47.79 A N ATOM 995 CARG A 416 44.539 22.452 41.954 1.00 14.71 A C ATOM 996 O ARG A 41644.260 23.636 41.819 1.00 17.79 A O ATOM 997 N LEU A 417 44.029 21.50941.173 1.00 15.18 A N ATOM 998 CA LEU A 417 43.092 21.820 40.099 1.0016.31 A C ATOM 999 CB LEU A 417 42.602 20.533 39.445 1.00 17.35 A C ATOM1000 CG LEU A 417 43.236 20.062 38.146 1.00 21.89 A C ATOM 1001 CD1 LEUA 417 44.676 19.654 38.383 1.00 21.71 A C ATOM 1002 CD2 LEU A 417 42.42218.910 37.580 1.00 19.02 A C ATOM 1003 C LEU A 417 41.864 22.559 40.6041.00 22.00 A C ATOM 1004 O LEU A 417 41.407 22.331 41.724 1.00 21.62 A OATOM 1005 N GLU A 418 41.319 23.437 39.771 1.00 15.35 A N ATOM 1006 CAGLU A 418 39.939 23.841 39.961 1.00 16.35 A C ATOM 1007 CB GLU A 41839.744 25.318 39.699 1.00 19.89 A C ATOM 1008 CG GLU A 418 40.727 26.22740.377 1.00 27.23 A C ATOM 1009 CD GLU A 418 40.787 27.541 39.662 1.0033.21 A C ATOM 1010 OE1 GLU A 418 40.238 27.607 38.528 1.00 30.20 A OATOM 1011 OE2 GLU A 418 41.378 28.490 40.217 1.00 45.03 A O ATOM 1012 CGLU A 418 39.096 23.053 38.975 1.00 15.57 A C ATOM 1013 O GLU A 41839.609 22.506 37.994 1.00 12.20 A O ATOM 1014 N ARG A 419 37.796 23.02439.226 1.00 12.89 A N ATOM 1015 CA ARG A 419 36.872 22.241 38.422 1.0012.88 A C ATOM 1016 CB ARG A 419 35.471 22.342 39.028 1.00 12.88 A CATOM 1017 CG ARG A 419 34.466 21.368 38.453 1.00 24.19 A C ATOM 1018 CDARG A 419 33.216 21.347 39.314 1.00 24.82 A C ATOM 1019 NE ARG A 41932.239 20.377 38.828 1.00 36.72 A N ATOM 1020 CZ ARG A 419 32.388 19.05738.903 1.00 37.97 A C ATOM 1021 NH1 ARG A 419 33.485 18.527 39.439 1.0033.99 A N ATOM 1022 NH2 ARG A 419 31.435 18.262 38.437 1.00 38.54 A NATOM 1023 C ARG A 419 36.874 22.669 36.944 1.00 12.43 A C ATOM 1024 OARG A 419 36.760 21.829 36.047 1.00 11.81 A O ATOM 1025 N GLU A 42037.011 23.971 36.697 1.00 9.29 A N ATOM 1026 CA GLU A 420 37.087 24.50235.333 1.00 8.71 A C ATOM 1027 CB GLU A 420 37.185 26.027 35.346 1.008.84 A C ATOM 1028 CG GLU A 420 35.932 26.745 35.786 1.00 8.55 A C ATOM1029 CD GLU A 420 35.794 26.835 37.297 1.00 12.59 A C ATOM 1030 OE1 GLUA 420 34.795 27.428 37.761 1.00 16.63 A O ATOM 1031 OE2 GLU A 420 36.67026.324 38.020 1.00 11.37 A O ATOM 1032 C GLU A 420 38.287 23.935 34.5711.00 10.65 A C ATOM 1033 O GLU A 420 38.205 23.643 33.371 1.00 8.02 A OATOM 1034 N GLU A 421 39.409 23.800 35.272 1.00 10.35 A N ATOM 1035 CAGLU A 421 40.617 23.285 34.653 1.00 9.75 A C ATOM 1036 CB GLU A 42141.802 23.492 35.586 1.00 8.77 A C ATOM 1037 CG GLU A 421 42.093 24.95335.830 1.00 11.31 A C ATOM 1038 CD GLU A 421 43.241 25.177 36.782 1.0013.29 A C ATOM 1039 OE1 GLU A 421 43.246 24.571 37.875 1.00 13.11 A OATOM 1040 OE2 GLU A 421 44.136 25.972 36.438 1.00 11.52 A O ATOM 1041 CGLU A 421 40.434 21.807 34.302 1.00 8.84 A C ATOM 1042 O GLU A 42140.802 21.366 33.213 1.00 11.01 A O ATOM 1043 N TYR A 422 39.856 21.05535.226 1.00 8.23 A N ATOM 1044 CA TYR A 422 39.553 19.650 34.995 1.008.38 A C ATOM 1045 CB TYR A 422 38.929 19.036 36.251 1.00 10.40 A C ATOM1046 CG TYR A 422 38.035 17.840 36.016 1.00 9.57 A C ATOM 1047 CD1 TYR A422 38.563 16.613 35.622 1.00 8.81 A C ATOM 1048 CE1 TYR A 422 37.73115.497 35.426 1.00 8.70 A C ATOM 1049 CD2 TYR A 422 36.660 17.927 36.2351.00 10.68 A C ATOM 1050 CE2 TYR A 422 35.826 16.829 36.050 1.00 13.62 AC ATOM 1051 CZ TYR A 422 36.363 15.616 35.646 1.00 12.55 A C ATOM 1052OH TYR A 422 35.521 14.530 35.464 1.00 11.27 A O ATOM 1053 C TYR A 42238.681 19.425 33.751 1.00 7.77 A C ATOM 1054 O TYR A 422 39.045 18.64532.881 1.00 9.57 A O ATOM 1055 N VAL A 423 37.546 20.109 33.633 1.006.84 A N ATOM 1056 CA VAL A 423 36.706 19.885 32.453 1.00 7.87 A C ATOM1057 CB VAL A 423 35.308 20.520 32.570 1.00 9.33 A C ATOM 1058 CG1 VAL A423 34.537 19.908 33.750 1.00 10.97 A C ATOM 1059 CG2 VAL A 423 35.40622.043 32.710 1.00 8.96 A C ATOM 1060 C VAL A 423 37.402 20.319 31.1521.00 7.11 A C ATOM 1061 O VAL A 423 37.269 19.663 30.120 1.00 6.06 A OATOM 1062 N LEU A 424 38.168 21.401 31.196 1.00 6.15 A N ATOM 1063 CALEU A 424 38.894 21.818 30.002 1.00 6.24 A C ATOM 1064 CB LEU A 42439.511 23.210 30.175 1.00 6.57 A C ATOM 1065 CG LEU A 424 38.531 24.38930.072 1.00 8.76 A C ATOM 1066 CD1 LEU A 424 39.009 25.609 30.869 1.009.27 A C ATOM 1067 CD2 LEU A 424 38.270 24.763 28.620 1.00 9.39 A C ATOM1068 C LEU A 424 39.954 20.772 29.644 1.00 8.74 A C ATOM 1069 O LEU A424 40.070 20.355 28.486 1.00 5.94 A O ATOM 1070 N LEU A 425 40.70120.328 30.651 1.00 6.82 A N ATOM 1071 CA LEU A 425 41.749 19.342 30.4411.00 5.92 A C ATOM 1072 CB LEU A 425 42.475 19.065 31.748 1.00 6.23 A CATOM 1073 CG LEU A 425 43.680 18.124 31.646 1.00 6.80 A C ATOM 1074 CD1LEU A 425 44.758 18.748 30.761 1.00 6.80 A C ATOM 1075 CD2 LEU A 42544.221 17.830 33.026 1.00 6.21 A C ATOM 1076 C LEU A 425 41.160 18.03629.917 1.00 7.43 A C ATOM 1077 O LEU A 425 41.731 17.387 29.031 1.005.47 A O ATOM 1078 N LYS A 426 40.017 17.648 30.475 1.00 5.57 A N ATOM1079 CA LYS A 426 39.379 16.406 30.067 1.00 7.09 A C ATOM 1080 CB LYS A426 38.214 16.070 31.001 1.00 7.82 A C ATOM 1081 CG LYS A 426 37.47814.810 30.614 1.00 6.77 A C ATOM 1082 CD LYS A 426 36.706 14.267 31.7891.00 11.38 A C ATOM 1083 CE LYS A 426 35.480 15.104 32.087 1.00 9.60 A CATOM 1084 NZ LYS A 426 34.510 14.302 32.900 1.00 14.46 A N ATOM 1085 CLYS A 426 38.919 16.462 28.609 1.00 6.38 A C ATOM 1086 O LYS A 42639.111 15.515 27.841 1.00 7.93 A O ATOM 1087 N ALA A 427 38.307 17.57528.229 1.00 7.50 A N ATOM 1088 CA ALA A 427 37.853 17.762 26.856 1.008.16 A C ATOM 1089 CB ALA A 427 37.090 19.081 26.719 1.00 5.44 A C ATOM1090 C ALA A 427 39.053 17.730 25.921 1.00 7.13 A C ATOM 1091 O ALA A427 39.004 17.133 24.842 1.00 5.94 A O ATOM 1092 N LEU A 428 40.13918.361 26.360 1.00 7.29 A N ATOM 1093 CA LEU A 428 41.380 18.398 25.5891.00 8.01 A C ATOM 1094 CB LEU A 428 42.397 19.339 26.245 1.00 6.63 A CATOM 1095 CG LEU A 428 43.703 19.529 25.472 1.00 8.24 A C ATOM 1096 CD1LEU A 428 43.417 20.011 24.046 1.00 9.06 A C ATOM 1097 CD2 LEU A 42844.630 20.502 26.213 1.00 7.30 A C ATOM 1098 C LEU A 428 41.977 17.00525.430 1.00 6.79 A C ATOM 1099 O LEU A 428 42.481 16.658 24.358 1.006.14 A O ATOM 1100 N ALA A 429 41.912 16.200 26.487 1.00 7.19 A N ATOM1101 CA ALA A 429 42.415 14.836 26.411 1.00 5.65 A C ATOM 1102 CB ALA A429 42.320 14.157 27.770 1.00 6.20 A C ATOM 1103 C ALA A 429 41.65014.037 25.360 1.00 6.78 A C ATOM 1104 O ALA A 429 42.214 13.199 24.6661.00 5.45 A O ATOM 1105 N LEU A 430 40.344 14.270 25.279 1.00 7.60 A NATOM 1106 CA LEU A 430 39.530 13.624 24.264 1.00 8.00 A C ATOM 1107 CBLEU A 430 38.082 14.077 24.391 1.00 5.99 A C ATOM 1108 CG LEU A 43037.151 13.694 23.237 1.00 10.42 A C ATOM 1109 CD1 LEU A 430 36.83712.206 23.290 1.00 8.94 A C ATOM 1110 CD2 LEU A 430 35.858 14.522 23.2951.00 10.83 A C ATOM 1111 C LEU A 430 40.046 13.986 22.876 1.00 8.39 A CATOM 1112 O LEU A 430 40.214 13.114 22.019 1.00 7.76 A O ATOM 1113 N ALAA 431 40.307 15.276 22.672 1.00 5.46 A N ATOM 1114 CA ALA A 431 40.66815.787 21.357 1.00 7.20 A C ATOM 1115 CB ALA A 431 40.371 17.277 21.2691.00 7.30 A C ATOM 1116 C ALA A 431 42.126 15.534 21.032 1.00 6.17 A CATOM 1117 O ALA A 431 42.556 15.760 19.911 1.00 7.37 A O ATOM 1118 N ASNA 432 42.891 15.084 22.019 1.00 6.25 A N ATOM 1119 CA ASN A 432 44.30614.828 21.787 1.00 5.84 A C ATOM 1120 CB ASN A 432 45.172 15.339 22.9341.00 5.42 A C ATOM 1121 CG ASN A 432 46.630 15.449 22.532 1.00 7.14 A CATOM 1122 OD1 ASN A 432 46.932 15.864 21.414 1.00 7.76 A O ATOM 1123 ND2ASN A 432 47.532 15.064 23.421 1.00 4.33 A N ATOM 1124 C ASN A 43244.593 13.355 21.518 1.00 5.85 A C ATOM 1125 O ASN A 432 45.739 12.90021.620 1.00 5.13 A O ATOM 1126 N SER A 433 43.535 12.622 21.192 1.006.01 A N ATOM 1127 CA SER A 433 43.640 11.238 20.740 1.00 8.50 A C ATOM1128 CB SER A 433 42.285 10.768 20.217 1.00 7.80 A C ATOM 1129 OG SER A433 42.441 9.759 19.228 1.00 10.95 A O ATOM 1130 C SER A 433 44.67611.075 19.630 1.00 8.64 A C ATOM 1131 O SER A 433 44.811 11.936 18.7621.00 9.43 A O ATOM 1132 N ASP A 434 45.395 9.959 19.653 1.00 6.22 A NATOM 1133 CA ASP A 434 46.324 9.643 18.584 1.00 9.48 A C ATOM 1134 CBASP A 434 47.749 9.555 19.131 1.00 7.92 A C ATOM 1135 CG ASP A 43448.320 10.924 19.465 1.00 7.14 A C ATOM 1136 OD1 ASP A 434 48.600 11.18620.660 1.00 5.12 A O ATOM 1137 OD2 ASP A 434 48.465 11.740 18.526 1.007.55 A O ATOM 1138 C ASP A 434 45.925 8.346 17.888 1.00 18.02 A C ATOM1139 O ASP A 434 46.773 7.536 17.516 1.00 19.74 A O ATOM 1140 N SER A435 44.623 8.148 17.721 1.00 17.08 A N ATOM 1141 CA SER A 435 44.1256.960 17.038 1.00 17.65 A C ATOM 1142 CB SER A 435 42.602 7.017 16.9051.00 18.23 A C ATOM 1143 OG SER A 435 42.142 6.004 16.025 1.00 23.32 A OATOM 1144 C SER A 435 44.747 6.830 15.653 1.00 19.96 A C ATOM 1145 O SERA 435 44.768 7.787 14.876 1.00 14.76 A O ATOM 1146 N VAL A 436 45.2315.631 15.347 1.00 25.14 A N ATOM 1147 CA VAL A 436 45.766 5.313 14.0281.00 24.59 A C ATOM 1148 CB VAL A 436 46.382 3.908 14.030 1.00 25.48 A CATOM 1149 CG1 VAL A 436 46.627 3.462 15.461 1.00 31.74 A C ATOM 1150 CG2VAL A 436 45.454 2.927 13.349 1.00 29.48 A C ATOM 1151 C VAL A 43644.687 5.364 12.935 1.00 24.11 A C ATOM 1152 O VAL A 436 44.997 5.48411.747 1.00 27.48 A O ATOM 1153 N HIS A 437 43.423 5.286 13.341 1.0023.04 A N ATOM 1154 CA HIS A 437 42.313 5.168 12.389 1.00 24.50 A C ATOM1155 CB HIS A 437 41.287 4.159 12.904 1.00 22.95 A C ATOM 1156 CG HIS A437 41.883 2.827 13.222 1.00 24.70 A C ATOM 1157 CD2 HIS A 437 42.0962.204 14.405 1.00 27.02 A C ATOM 1158 ND1 HIS A 437 42.376 1.987 12.2491.00 28.99 A N ATOM 1159 CE1 HIS A 437 42.850 0.891 12.817 1.00 35.30 AC ATOM 1160 NE2 HIS A 437 42.696 1.000 14.124 1.00 32.58 A N ATOM 1161 CHIS A 437 41.610 6.473 12.050 1.00 25.58 A C ATOM 1162 O HIS A 43740.536 6.460 11.450 1.00 22.32 A O ATOM 1163 N ILE A 438 42.202 7.59712.432 1.00 19.92 A N ATOM 1164 CA ILE A 438 41.607 8.892 12.119 1.0020.49 A C ATOM 1165 CB ILE A 438 42.228 10.005 12.982 1.00 17.72 A CATOM 1166 CG2 ILE A 438 41.957 11.381 12.366 1.00 19.00 A C ATOM 1167CG1 ILE A 438 41.699 9.913 14.421 1.00 16.43 A C ATOM 1168 CD1 ILE A 43842.615 10.586 15.455 1.00 18.54 A C ATOM 1169 C ILE A 438 41.764 9.21810.632 1.00 22.78 A C ATOM 1170 O ILE A 438 42.867 9.151 10.091 1.0026.18 A O ATOM 1171 N GLU A 439 40.666 9.570 9.970 1.00 21.53 A N ATOM1172 CA GLU A 439 40.713 9.815 8.533 1.00 22.55 A C ATOM 1173 CB GLU A439 39.501 9.194 7.836 1.00 25.78 A C ATOM 1174 CG GLU A 439 38.1689.888 8.108 1.00 33.70 A C ATOM 1175 CD GLU A 439 36.984 9.020 7.6931.00 40.85 A C ATOM 1176 OE1 GLU A 439 35.822 9.469 7.838 1.00 31.28 A OATOM 1177 OE2 GLU A 439 37.227 7.882 7.227 1.00 34.29 A O ATOM 1178 CGLU A 439 40.868 11.291 8.159 1.00 23.80 A C ATOM 1179 O GLU A 43941.462 11.614 7.133 1.00 23.54 A O ATOM 1180 N ASP A 440 40.334 12.1858.984 1.00 24.35 A N ATOM 1181 CA ASP A 440 40.566 13.612 8.792 1.0024.76 A C ATOM 1182 CB ASP A 440 39.250 14.377 8.680 1.00 20.26 A C ATOM1183 CG ASP A 440 39.440 15.771 8.124 1.00 23.85 A C ATOM 1184 OD1 ASP A440 40.601 16.240 8.067 1.00 26.74 A O ATOM 1185 OD2 ASP A 440 38.42916.396 7.740 1.00 27.40 A O ATOM 1186 C ASP A 440 41.423 14.181 9.9221.00 22.00 A C ATOM 1187 O ASP A 440 40.906 14.710 10.910 1.00 18.34 A OATOM 1188 N ALA A 441 42.734 14.073 9.755 1.00 17.84 A N ATOM 1189 CAALA A 441 43.681 14.430 10.805 1.00 22.82 A C ATOM 1190 CB ALA A 44145.080 13.929 10.454 1.00 20.64 A C ATOM 1191 C ALA A 441 43.713 15.92611.099 1.00 21.00 A C ATOM 1192 O ALA A 441 44.000 16.338 12.228 1.0019.72 A O ATOM 1193 N GLU A 442 43.437 16.749 10.095 1.00 21.58 A N ATOM1194 CA GLU A 442 43.464 18.184 10.343 1.00 20.04 A C ATOM 1195 CB GLU A442 43.779 18.984 9.073 1.00 25.38 A C ATOM 1196 CG GLU A 442 42.63319.175 8.106 1.00 39.10 A C ATOM 1197 CD GLU A 442 43.007 20.112 6.9591.00 54.49 A C ATOM 1198 OE1 GLU A 442 43.856 19.725 6.123 1.00 61.64 AO ATOM 1199 OE2 GLU A 442 42.457 21.235 6.898 1.00 47.00 A O ATOM 1200 CGLU A 442 42.176 18.636 11.029 1.00 19.84 A C ATOM 1201 O GLU A 44242.169 19.609 11.775 1.00 19.40 A O ATOM 1202 N ALA A 443 41.091 17.90810.796 1.00 17.15 A N ATOM 1203 CA ALA A 443 39.840 18.207 11.476 1.0018.23 A C ATOM 1204 CB ALA A 443 38.725 17.378 10.911 1.00 15.37 A CATOM 1205 C ALA A 443 39.991 17.945 12.974 1.00 18.94 A C ATOM 1206 OALA A 443 39.504 18.720 13.800 1.00 15.50 A O ATOM 1207 N VAL A 44440.651 16.842 13.317 1.00 11.01 A N ATOM 1208 CA VAL A 444 40.915 16.53414.714 1.00 14.83 A C ATOM 1209 CB VAL A 444 41.528 15.136 14.903 1.0012.93 A C ATOM 1210 CG1 VAL A 444 41.880 14.916 16.372 1.00 12.78 A CATOM 1211 CG2 VAL A 444 40.586 14.064 14.402 1.00 9.54 A C ATOM 1212 CVAL A 444 41.901 17.546 15.264 1.00 12.87 A C ATOM 1213 O VAL A 44441.746 18.028 16.380 1.00 9.39 A O ATOM 1214 N GLU A 445 42.924 17.85914.475 1.00 12.91 A N ATOM 1215 CA GLU A 445 43.889 18.861 14.891 1.0014.38 A C ATOM 1216 CB GLU A 445 44.917 19.134 13.797 1.00 15.55 A CATOM 1217 CG GLU A 445 45.958 20.144 14.205 1.00 16.05 A C ATOM 1218 CDGLU A 445 46.977 20.408 13.117 1.00 22.16 A C ATOM 1219 OE1 GLU A 44548.173 20.152 13.352 1.00 22.61 A O ATOM 1220 OE2 GLU A 445 46.58520.874 12.026 1.00 30.96 A O ATOM 1221 C GLU A 445 43.177 20.159 15.2631.00 14.87 A C ATOM 1222 O GLU A 445 43.474 20.756 16.291 1.00 11.42 A OATOM 1223 N GLN A 446 42.220 20.585 14.440 1.00 15.11 A N ATOM 1224 CAGLN A 446 41.568 21.860 14.695 1.00 17.36 A C ATOM 1225 CB GLN A 44640.759 22.335 13.482 1.00 17.05 A C ATOM 1226 CG GLN A 446 39.275 22.05813.571 1.00 31.52 A C ATOM 1227 CD GLN A 446 38.458 22.993 12.690 1.0046.25 A C ATOM 1228 OE1 GLN A 446 38.992 23.634 11.774 1.00 40.21 A OATOM 1229 NE2 GLN A 446 37.153 23.077 12.965 1.00 42.91 A N ATOM 1230 CGLN A 446 40.729 21.825 15.983 1.00 14.65 A C ATOM 1231 O GLN A 44640.694 22.800 16.732 1.00 11.67 A O ATOM 1232 N LEU A 447 40.068 20.70216.251 1.00 15.45 A N ATOM 1233 CA LEU A 447 39.338 20.556 17.511 1.0011.38 A C ATOM 1234 CB LEU A 447 38.572 19.232 17.555 1.00 11.90 A CATOM 1235 CG LEU A 447 37.819 18.964 18.863 1.00 13.32 A C ATOM 1236 CD1LEU A 447 36.859 20.108 19.173 1.00 13.67 A C ATOM 1237 CD2 LEU A 44737.072 17.637 18.795 1.00 10.35 A C ATOM 1238 C LEU A 447 40.298 20.64418.700 1.00 12.43 A C ATOM 1239 O LEU A 447 40.036 21.361 19.667 1.0010.76 A O ATOM 1240 N ARG A 448 41.407 19.906 18.619 1.00 8.12 A N ATOM1241 CA ARG A 448 42.438 19.912 19.664 1.00 8.60 A C ATOM 1242 CB ARG A448 43.599 19.002 19.264 1.00 9.77 A C ATOM 1243 CG ARG A 448 44.71018.863 20.299 1.00 7.47 A C ATOM 1244 CD ARG A 448 46.029 18.548 19.6001.00 6.92 A C ATOM 1245 NE ARG A 448 45.902 17.399 18.710 1.00 11.60 A NATOM 1246 CZ ARG A 448 46.486 17.295 17.517 1.00 12.15 A C ATOM 1247 NH1ARG A 448 47.233 18.285 17.030 1.00 10.50 A N ATOM 1248 NH2 ARG A 44846.305 16.203 16.795 1.00 9.94 A N ATOM 1249 C ARG A 448 42.965 21.32819.893 1.00 13.39 A C ATOM 1250 O ARG A 448 43.060 21.790 21.034 1.0013.33 A O ATOM 1251 N GLU A 449 43.305 22.019 18.805 1.00 9.29 A N ATOM1252 CA GLU A 449 43.770 23.403 18.904 1.00 13.30 A C ATOM 1253 CB GLU A449 44.149 23.959 17.526 1.00 12.75 A C ATOM 1254 CG GLU A 449 45.54923.575 17.075 1.00 15.91 A C ATOM 1255 CD GLU A 449 45.780 23.844 15.5991.00 22.96 A C ATOM 1256 OE1 GLU A 449 44.906 24.470 14.956 1.00 24.52 AO ATOM 1257 OE2 GLU A 449 46.831 23.420 15.076 1.00 17.98 A O ATOM 1258C GLU A 449 42.758 24.326 19.579 1.00 10.98 A C ATOM 1259 O GLU A 44943.137 25.171 20.393 1.00 10.65 A O ATOM 1260 N ALA A 450 41.480 24.17519.233 1.00 8.22 A N ATOM 1261 CA ALA A 450 40.443 25.042 19.779 1.009.95 A C ATOM 1262 CB ALA A 450 39.093 24.815 19.062 1.00 9.97 A C ATOM1263 C ALA A 450 40.288 24.828 21.277 1.00 10.41 A C ATOM 1264 O ALA A450 40.073 25.778 22.022 1.00 11.91 A O ATOM 1265 N LEU A 451 40.37923.579 21.723 1.00 10.66 A N ATOM 1266 CA LEU A 451 40.256 23.296 23.1511.00 7.86 A C ATOM 1267 CB LEU A 451 39.934 21.824 23.389 1.00 9.13 A CATOM 1268 CG LEU A 451 38.504 21.519 22.934 1.00 8.28 A C ATOM 1269 CD1LEU A 451 38.276 20.030 22.816 1.00 11.12 A C ATOM 1270 CD2 LEU A 45137.507 22.162 23.895 1.00 6.01 A C ATOM 1271 C LEU A 451 41.492 23.73623.926 1.00 10.55 A C ATOM 1272 O LEU A 451 41.385 24.208 25.058 1.0010.13 A O ATOM 1273 N HIS A 452 42.664 23.594 23.310 1.00 7.71 A N ATOM1274 CA HIS A 452 43.884 24.095 23.917 1.00 11.17 A C ATOM 1275 CB HIS A452 45.099 23.727 23.064 1.00 9.18 A C ATOM 1276 CG HIS A 452 46.39624.236 23.603 1.00 10.33 A C ATOM 1277 CD2 HIS A 452 46.807 24.46224.874 1.00 11.74 A C ATOM 1278 ND1 HIS A 452 47.453 24.585 22.789 1.0013.92 A N ATOM 1279 CE1 HIS A 452 48.464 24.997 23.538 1.00 13.80 A CATOM 1280 NE2 HIS A 452 48.099 24.933 24.804 1.00 12.48 A N ATOM 1281 CHIS A 452 43.787 25.615 24.099 1.00 10.62 A C ATOM 1282 O HIS A 45244.086 26.132 25.174 1.00 10.48 A O ATOM 1283 N GLU A 453 43.362 26.32423.055 1.00 11.13 A N ATOM 1284 CA GLU A 453 43.245 27.783 23.135 1.0013.13 A C ATOM 1285 CB GLU A 453 42.901 28.386 21.768 1.00 15.88 A CATOM 1286 CG GLU A 453 42.436 29.856 21.810 1.00 26.46 A C ATOM 1287 CDGLU A 453 43.584 30.868 21.927 1.00 37.28 A C ATOM 1288 OE1 GLU A 45343.306 32.047 22.253 1.00 40.62 A O ATOM 1289 OE2 GLU A 453 44.75830.491 21.693 1.00 33.52 A O ATOM 1290 C GLU A 453 42.214 28.185 24.1931.00 12.79 A C ATOM 1291 O GLU A 453 42.394 29.172 24.904 1.00 9.59 A OATOM 1292 N ALA A 454 41.139 27.404 24.301 1.00 10.51 A N ATOM 1293 CAALA A 454 40.165 27.600 25.366 1.00 10.72 A C ATOM 1294 CB ALA A 45439.044 26.560 25.273 1.00 9.25 A C ATOM 1295 C ALA A 454 40.846 27.53426.732 1.00 10.67 A C ATOM 1296 O ALA A 454 40.652 28.403 27.568 1.0010.84 A O ATOM 1297 N LEU A 455 41.640 26.492 26.964 1.00 10.88 A N ATOM1298 CA LEU A 455 42.345 26.353 28.236 1.00 8.61 A C ATOM 1299 CB LEU A455 43.221 25.088 28.250 1.00 7.42 A C ATOM 1300 CG LEU A 455 44.12724.857 29.472 1.00 8.75 A C ATOM 1301 CD1 LEU A 455 43.341 24.897 30.7821.00 6.63 A C ATOM 1302 CD2 LEU A 455 44.913 23.547 29.352 1.00 6.89 A CATOM 1303 C LEU A 455 43.200 27.588 28.504 1.00 9.40 A C ATOM 1304 O LEUA 455 43.184 28.138 29.607 1.00 7.99 A O ATOM 1305 N LEU A 456 43.95028.012 27.487 1.00 10.89 A N ATOM 1306 CA LEU A 456 44.818 29.186 27.5831.00 11.72 A C ATOM 1307 CB LEU A 456 45.549 29.396 26.254 1.00 14.86 AC ATOM 1308 CG LEU A 456 47.032 29.041 26.111 1.00 21.47 A C ATOM 1309CD1 LEU A 456 47.549 28.219 27.269 1.00 14.74 A C ATOM 1310 CD2 LEU A456 47.300 28.348 24.772 1.00 17.27 A C ATOM 1311 C LEU A 456 44.02830.450 27.921 1.00 12.19 A C ATOM 1312 O LEU A 456 44.414 31.229 28.8001.00 10.93 A O ATOM 1313 N GLU A 457 42.928 30.656 27.202 1.00 9.66 A NATOM 1314 CA GLU A 457 42.117 31.845 27.399 1.00 11.10 A C ATOM 1315 CBGLU A 457 41.012 31.938 26.348 1.00 13.16 A C ATOM 1316 CG GLU A 45741.526 32.147 24.927 1.00 16.72 A C ATOM 1317 CD GLU A 457 40.403 32.20523.888 1.00 29.59 A C ATOM 1318 OE1 GLU A 457 40.717 32.236 22.676 1.0030.94 A O ATOM 1319 OE2 GLU A 457 39.213 32.220 24.280 1.00 26.23 A OATOM 1320 C GLU A 457 41.523 31.823 28.795 1.00 11.37 A C ATOM 1321 OGLU A 457 41.504 32.846 29.478 1.00 11.20 A O ATOM 1322 N TYR A 45841.058 30.652 29.230 1.00 9.53 A N ATOM 1323 CA TYR A 458 40.542 30.53630.591 1.00 14.47 A C ATOM 1324 CB TYR A 458 40.051 29.121 30.928 1.0010.65 A C ATOM 1325 CG TYR A 458 39.702 29.013 32.400 1.00 14.28 A CATOM 1326 CD1 TYR A 458 38.490 29.501 32.891 1.00 13.02 A C ATOM 1327CE1 TYR A 458 38.179 29.425 34.242 1.00 16.48 A C ATOM 1328 CD2 TYR A458 40.602 28.469 33.305 1.00 14.10 A C ATOM 1329 CE2 TYR A 458 40.30528.393 34.654 1.00 16.20 A C ATOM 1330 CZ TYR A 458 39.095 28.872 35.1171.00 18.51 A C ATOM 1331 OH TYR A 458 38.811 28.781 36.460 1.00 20.92 AO ATOM 1332 C TYR A 458 41.598 30.955 31.607 1.00 15.93 A C ATOM 1333 OTYR A 458 41.324 31.750 32.514 1.00 14.40 A O ATOM 1334 N GLU A 45942.807 30.422 31.455 1.00 11.63 A N ATOM 1335 CA GLU A 459 43.875 30.71632.401 1.00 13.04 A C ATOM 1336 CB GLU A 459 45.127 29.892 32.094 1.0015.32 A C ATOM 1337 CG GLU A 459 44.930 28.394 32.295 1.00 12.08 A CATOM 1338 CD GLU A 459 44.667 28.034 33.745 1.00 13.32 A C ATOM 1339 OE1GLU A 459 44.650 28.942 34.606 1.00 15.61 A O ATOM 1340 OE2 GLU A 45944.484 26.841 34.033 1.00 10.12 A O ATOM 1341 C GLU A 459 44.208 32.20032.386 1.00 16.76 A C ATOM 1342 O GLU A 459 44.403 32.812 33.439 1.0020.81 A O ATOM 1343 N ALA A 460 44.267 32.772 31.189 1.00 13.37 A N ATOM1344 CA ALA A 460 44.658 34.167 31.022 1.00 18.22 A C ATOM 1345 CB ALA A460 44.771 34.518 29.530 1.00 17.66 A C ATOM 1346 C ALA A 460 43.66735.096 31.713 1.00 18.87 A C ATOM 1347 O ALA A 460 44.027 36.187 32.1551.00 17.28 A O ATOM 1348 N GLY A 461 42.420 34.651 31.811 1.00 13.96 A NATOM 1349 CA GLY A 461 41.367 35.480 32.359 1.00 17.50 A C ATOM 1350 CGLY A 461 41.030 35.266 33.824 1.00 24.82 A C ATOM 1351 O GLY A 46140.097 35.888 34.326 1.00 24.71 A O ATOM 1352 N ARG A 462 41.761 34.39234.516 1.00 27.99 A N ATOM 1353 CA ARG A 462 41.496 34.184 35.939 1.0027.80 A C ATOM 1354 CB ARG A 462 42.321 33.033 36.529 1.00 27.47 A CATOM 1355 CG ARG A 462 42.322 31.756 35.713 1.00 28.09 A C ATOM 1356 CDARG A 462 41.902 30.549 36.550 1.00 30.71 A C ATOM 1357 NE ARG A 46242.527 30.494 37.870 1.00 30.04 A N ATOM 1358 CZ ARG A 462 43.352 29.52838.270 1.00 38.66 A C ATOM 1359 NH1 ARG A 462 43.661 28.534 37.447 1.0027.12 A N ATOM 1360 NH2 ARG A 462 43.870 29.553 39.495 1.00 38.79 A NATOM 1361 C ARG A 462 41.781 35.475 36.703 1.00 30.36 A C ATOM 1362 OARG A 462 40.864 36.121 37.215 1.00 33.54 A O ATOM 1363 N GLY A 46757.554 33.642 29.449 1.00 31.18 N ATOM 1364 CA GLY A 467 57.315 32.74130.563 1.00 33.18 C ATOM 1365 C GLY A 467 55.889 32.770 31.090 1.0043.87 C ATOM 1366 O GLY A 467 55.650 32.403 32.246 1.00 49.22 O ATOM1367 N GLY A 468 54.950 33.187 30.235 1.00 44.36 N ATOM 1368 CA GLY A468 53.550 33.393 30.594 1.00 38.41 C ATOM 1369 C GLY A 468 53.03432.617 31.790 1.00 43.19 C ATOM 1370 O GLY A 468 53.016 31.399 31.7611.00 42.24 O ATOM 1371 O ALA A 469 50.412 31.103 34.683 1.00 38.34 OATOM 1372 N ALA A 469 52.606 33.327 32.837 1.00 57.57 N ATOM 1373 CA ALAA 469 52.127 32.699 34.077 1.00 47.79 C ATOM 1374 C ALA A 469 50.86231.883 33.831 1.00 42.70 C ATOM 1375 CB ALA A 469 51.886 33.747 35.1521.00 53.53 C ATOM 1376 N GLU A 470 50.286 32.077 32.652 1.00 49.90 NATOM 1377 CA GLU A 470 49.229 31.206 32.173 1.00 44.12 C ATOM 1378 C GLUA 470 49.866 29.881 31.772 1.00 38.68 C ATOM 1379 CB GLU A 470 48.51731.848 30.987 1.00 45.04 C ATOM 1380 CG GLU A 470 48.080 33.273 31.2721.00 37.73 C ATOM 1381 CD GLU A 470 48.604 34.255 30.246 1.00 45.69 CATOM 1382 OE1 GLU A 470 47.821 34.670 29.364 1.00 45.18 O ATOM 1383 OE2GLU A 470 49.805 34.606 30.316 1.00 53.29 O ATOM 1384 O GLU A 470 49.18729.035 31.176 1.00 22.49 O ATOM 1385 N ARG A 472 51.175 29.768 32.0841.00 38.15 A N ATOM 1386 CA ARG A 472 52.000 28.538 32.163 1.00 28.98 AC ATOM 1387 CB ARG A 472 53.407 28.910 32.624 1.00 32.47 A C ATOM 1388CG ARG A 472 53.960 28.048 33.758 1.00 28.83 A C ATOM 1389 CD ARG A 47255.076 28.765 34.521 1.00 36.97 A C ATOM 1390 NE ARG A 472 54.552 29.76135.457 1.00 49.68 A N ATOM 1391 CZ ARG A 472 55.221 30.835 35.875 1.0048.56 A C ATOM 1392 NH1 ARG A 472 56.449 31.077 35.435 1.00 47.42 A NATOM 1393 NH2 ARG A 472 54.654 31.679 36.727 1.00 57.24 A N ATOM 1394 CARG A 472 51.460 27.528 33.167 1.00 22.66 A C ATOM 1395 O ARG A 47251.953 26.385 33.296 1.00 15.82 A O ATOM 1396 N ARG A 473 50.483 27.99233.924 1.00 12.06 A N ATOM 1397 CA ARG A 473 49.577 27.123 34.611 1.0013.43 A C ATOM 1398 CB ARG A 473 48.402 27.962 35.118 1.00 14.06 A CATOM 1399 CG ARG A 473 47.281 27.168 35.726 1.00 16.08 A C ATOM 1400 CDARG A 473 47.744 26.481 36.985 1.00 14.45 A C ATOM 1401 NE ARG A 47346.686 25.663 37.574 1.00 18.74 A N ATOM 1402 CZ ARG A 473 46.863 24.90738.651 1.00 18.61 A C ATOM 1403 NH1 ARG A 473 48.060 24.882 39.231 1.0013.30 A N ATOM 1404 NH2 ARG A 473 45.858 24.181 39.143 1.00 13.45 A NATOM 1405 C ARG A 473 49.103 26.068 33.599 1.00 9.75 A C ATOM 1406 O ARGA 473 49.060 24.886 33.908 1.00 9.07 A O ATOM 1407 N ALA A 474 48.76426.503 32.387 1.00 8.60 A N ATOM 1408 CA ALA A 474 48.233 25.602 31.3571.00 12.29 A C ATOM 1409 CB ALA A 474 47.836 26.372 30.101 1.00 7.83 A CATOM 1410 C ALA A 474 49.209 24.476 31.001 1.00 9.60 A C ATOM 1411 O ALAA 474 48.821 23.312 30.918 1.00 6.48 A O ATOM 1412 N GLY A 475 50.47424.825 30.791 1.00 8.47 A N ATOM 1413 CA GLY A 475 51.472 23.820 30.4781.00 6.70 A C ATOM 1414 C GLY A 475 51.596 22.777 31.579 1.00 7.84 A CATOM 1415 O GLY A 475 51.806 21.594 31.308 1.00 6.38 A O ATOM 1416 N ARGA 476 51.470 23.207 32.830 1.00 7.27 A N ATOM 1417 CA ARG A 476 51.62122.276 33.937 1.00 9.01 A C ATOM 1418 CB ARG A 476 51.709 23.000 35.2851.00 10.28 A C ATOM 1419 CG ARG A 476 53.021 23.745 35.462 1.00 14.06 AC ATOM 1420 CD ARG A 476 53.149 24.466 36.810 1.00 13.23 A C ATOM 1421NE ARG A 476 54.392 25.229 36.835 1.00 21.02 A N ATOM 1422 CZ ARG A 47654.731 26.093 37.788 1.00 28.20 A C ATOM 1423 NH1 ARG A 476 53.91726.303 38.812 1.00 24.66 A N ATOM 1424 NH2 ARG A 476 55.887 26.74237.716 1.00 27.48 A N ATOM 1425 C ARG A 476 50.502 21.243 33.937 1.008.35 A C ATOM 1426 O ARG A 476 50.720 20.076 34.296 1.00 6.16 A O ATOM1427 N LEU A 477 49.307 21.668 33.544 1.00 9.30 A N ATOM 1428 CA LEU A477 48.212 20.713 33.384 1.00 8.69 A C ATOM 1429 CB LEU A 477 46.88521.429 33.139 1.00 7.76 A C ATOM 1430 CG LEU A 477 46.513 22.531 34.1421.00 11.82 A C ATOM 1431 CD1 LEU A 477 45.216 23.218 33.737 1.00 11.76 AC ATOM 1432 CD2 LEU A 477 46.399 21.973 35.540 1.00 9.57 A C ATOM 1433 CLEU A 477 48.526 19.755 32.229 1.00 5.56 A C ATOM 1434 O LEU A 47748.350 18.551 32.362 1.00 6.17 A O ATOM 1435 N LEU A 478 49.007 20.29431.109 1.00 6.20 A N ATOM 1436 CA LEU A 478 49.321 19.463 29.939 1.007.90 A C ATOM 1437 CB LEU A 478 49.795 20.297 28.739 1.00 6.55 A C ATOM1438 CG LEU A 478 48.915 21.411 28.161 1.00 10.60 A C ATOM 1439 CD1 LEUA 478 49.393 21.834 26.750 1.00 6.64 A C ATOM 1440 CD2 LEU A 478 47.46520.999 28.127 1.00 6.74 A C ATOM 1441 C LEU A 478 50.369 18.406 30.2661.00 4.61 A C ATOM 1442 O LEU A 478 50.329 17.306 29.723 1.00 4.57 A OATOM 1443 N LEU A 479 51.283 18.730 31.174 1.00 3.82 A N ATOM 1444 CALEU A 479 52.391 17.824 31.474 1.00 6.73 A C ATOM 1445 CB LEU A 47953.524 18.546 32.203 1.00 3.69 A C ATOM 1446 CG LEU A 479 54.400 19.45731.336 1.00 6.62 A C ATOM 1447 CD1 LEU A 479 55.179 20.409 32.225 1.006.04 A C ATOM 1448 CD2 LEU A 479 55.350 18.640 30.461 1.00 6.42 A C ATOM1449 C LEU A 479 51.958 16.587 32.266 1.00 5.21 A C ATOM 1450 O LEU A479 52.767 15.681 32.488 1.00 5.37 A O ATOM 1451 N THR A 480 50.70016.568 32.705 1.00 4.49 A N ATOM 1452 CA THR A 480 50.142 15.393 33.3711.00 5.19 A C ATOM 1453 CB THR A 480 49.115 15.771 34.454 1.00 5.02 A CATOM 1454 OG1 THR A 480 47.881 16.129 33.828 1.00 3.89 A O ATOM 1455 CG2THR A 480 49.612 16.943 35.313 1.00 7.08 A C ATOM 1456 C THR A 48049.458 14.442 32.376 1.00 5.92 A C ATOM 1457 O THR A 480 49.067 13.32332.730 1.00 4.76 A O ATOM 1458 N LEU A 481 49.315 14.879 31.131 1.004.99 A N ATOM 1459 CA LEU A 481 48.643 14.053 30.132 1.00 6.38 A C ATOM1460 CB LEU A 481 48.323 14.876 28.879 1.00 4.09 A C ATOM 1461 CG LEU A481 47.263 15.959 29.112 1.00 6.36 A C ATOM 1462 CD1 LEU A 481 47.09916.855 27.885 1.00 4.83 A C ATOM 1463 CD2 LEU A 481 45.928 15.311 29.5031.00 6.78 A C ATOM 1464 C LEU A 481 49.419 12.765 29.787 1.00 6.72 A CATOM 1465 O LEU A 481 48.823 11.723 29.508 1.00 7.93 A O ATOM 1466 N PROA 482 50.753 12.834 29.782 1.00 6.60 A N ATOM 1467 CD PRO A 482 51.64814.000 29.669 1.00 4.04 A C ATOM 1468 CA PRO A 482 51.450 11.572 29.5291.00 6.31 A C ATOM 1469 CB PRO A 482 52.924 11.978 29.568 1.00 7.44 A CATOM 1470 CG PRO A 482 52.914 13.410 29.084 1.00 4.45 A C ATOM 1471 CPRO A 482 51.135 10.516 30.590 1.00 7.02 A C ATOM 1472 O PRO A 48250.852 9.372 30.233 1.00 5.79 A O ATOM 1473 N LEU A 483 51.167 10.88631.867 1.00 6.14 A N ATOM 1474 CA LEU A 483 50.840 9.928 32.915 1.007.66 A C ATOM 1475 CB LEU A 483 51.102 10.501 34.317 1.00 5.49 A C ATOM1476 CG LEU A 483 50.748 9.589 35.501 1.00 6.58 A C ATOM 1477 CD1 LEU A483 51.488 8.251 35.455 1.00 7.85 A C ATOM 1478 CD2 LEU A 483 50.98410.286 36.848 1.00 8.45 A C ATOM 1479 C LEU A 483 49.387 9.491 32.7841.00 6.34 A C ATOM 1480 O LEU A 483 49.050 8.338 33.068 1.00 6.26 A OATOM 1481 N LEU A 484 48.519 10.405 32.361 1.00 5.41 A N ATOM 1482 CALEU A 484 47.112 10.037 32.191 1.00 4.94 A C ATOM 1483 CB LEU A 48446.267 11.235 31.773 1.00 5.72 A C ATOM 1484 CG LEU A 484 44.838 10.87131.359 1.00 8.11 A C ATOM 1485 CD1 LEU A 484 44.121 10.158 32.516 1.007.42 A C ATOM 1486 CD2 LEU A 484 44.037 12.092 30.873 1.00 4.36 A C ATOM1487 C LEU A 484 46.958 8.894 31.179 1.00 8.10 A C ATOM 1488 O LEU A 48446.278 7.899 31.448 1.00 7.35 A O ATOM 1489 N ARG A 485 47.600 9.03430.019 1.00 8.09 A N ATOM 1490 CA ARG A 485 47.514 8.009 28.978 1.005.79 A C ATOM 1491 CB ARG A 485 48.158 8.485 27.668 1.00 5.19 A C ATOM1492 CG ARG A 485 47.974 7.494 26.509 1.00 8.17 A C ATOM 1493 CD ARG A485 48.363 8.079 25.151 1.00 7.22 A C ATOM 1494 NE ARG A 485 47.4559.129 24.704 1.00 8.05 A N ATOM 1495 CZ ARG A 485 47.585 9.771 23.5501.00 6.38 A C ATOM 1496 NH1 ARG A 485 48.588 9.466 22.733 1.00 9.41 A NATOM 1497 NH2 ARG A 485 46.727 10.718 23.213 1.00 6.28 A N ATOM 1498 CARG A 485 48.126 6.682 29.445 1.00 9.91 A C ATOM 1499 O ARG A 485 47.5325.610 29.246 1.00 8.36 A O ATOM 1500 N GLN A 486 49.297 6.766 30.0771.00 6.47 A N ATOM 1501 CA GLN A 486 49.983 5.605 30.642 1.00 7.15 A CATOM 1502 CB GLN A 486 51.280 6.043 31.325 1.00 8.50 A C ATOM 1503 CGGLN A 486 52.116 4.889 31.885 1.00 13.05 A C ATOM 1504 CD GLN A 48653.436 5.358 32.486 1.00 18.63 A C ATOM 1505 OE1 GLN A 486 54.385 4.58632.611 1.00 27.27 A O ATOM 1506 NE2 GLN A 486 53.498 6.622 32.857 1.007.54 A N ATOM 1507 C GLN A 486 49.105 4.868 31.662 1.00 8.58 A C ATOM1508 O GLN A 486 49.034 3.634 31.671 1.00 10.15 A O ATOM 1509 N THR A487 48.458 5.629 32.533 1.00 6.57 A N ATOM 1510 CA THR A 487 47.6015.041 33.554 1.00 6.03 A C ATOM 1511 CB THR A 487 47.020 6.102 34.5081.00 6.25 A C ATOM 1512 OG1 THR A 487 48.089 6.869 35.083 1.00 6.80 A OATOM 1513 CG2 THR A 487 46.222 5.451 35.628 1.00 6.92 A C ATOM 1514 CTHR A 487 46.474 4.262 32.885 1.00 8.30 A C ATOM 1515 O THR A 487 46.1883.136 33.284 1.00 8.08 A O ATOM 1516 N ALA A 488 45.838 4.859 31.8691.00 7.90 A N ATOM 1517 CA ALA A 488 44.778 4.172 31.121 1.00 7.61 A CATOM 1518 CB ALA A 488 44.158 5.094 30.086 1.00 6.25 A C ATOM 1519 C ALAA 488 45.268 2.874 30.462 1.00 9.70 A C ATOM 1520 O ALA A 488 44.5701.854 30.480 1.00 9.54 A O ATOM 1521 N GLY A 489 46.467 2.915 29.8811.00 9.15 A N ATOM 1522 CA GLY A 489 47.078 1.724 29.316 1.00 8.13 A CATOM 1523 C GLY A 489 47.277 0.653 30.375 1.00 12.71 A C ATOM 1524 O GLYA 489 47.060 −0.540 30.124 1.00 13.11 A O ATOM 1525 N LYS A 490 47.6901.077 31.567 1.00 10.81 A N ATOM 1526 CA LYS A 490 47.919 0.151 32.6741.00 11.63 A C ATOM 1527 CB LYS A 490 48.531 0.883 33.874 1.00 10.68 A CATOM 1528 CG LYS A 490 50.058 1.014 33.831 1.00 12.15 A C ATOM 1529 CDLYS A 490 50.520 2.108 34.780 1.00 18.49 A C ATOM 1530 CE LYS A 49051.784 1.723 35.538 1.00 27.57 A C ATOM 1531 NZ LYS A 490 52.910 1.33234.646 1.00 30.65 A N ATOM 1532 C LYS A 490 46.617 −0.539 33.080 1.0010.40 A C ATOM 1533 O LYS A 490 46.594 −1.740 33.334 1.00 9.67 A O ATOM1534 N VAL A 491 45.531 0.227 33.136 1.00 10.22 A N ATOM 1535 CA VAL A491 44.227 −0.336 33.474 1.00 9.82 A C ATOM 1536 CB VAL A 491 43.1430.759 33.568 1.00 12.35 A C ATOM 1537 CG1 VAL A 491 41.758 0.129 33.7061.00 8.81 A C ATOM 1538 CG2 VAL A 491 43.435 1.709 34.740 1.00 8.17 A CATOM 1539 C VAL A 491 43.815 −1.394 32.444 1.00 10.85 A C ATOM 1540 OVAL A 491 43.376 −2.493 32.802 1.00 10.60 A O ATOM 1541 N LEU A 49243.970 −1.065 31.166 1.00 9.78 A N ATOM 1542 CA LEU A 492 43.612 −1.99930.101 1.00 11.20 A C ATOM 1543 CB LEU A 492 43.727 −1.335 28.726 1.0012.15 A C ATOM 1544 CG LEU A 492 42.641 −0.300 28.433 1.00 11.13 A CATOM 1545 CD1 LEU A 492 43.057 0.656 27.331 1.00 14.47 A C ATOM 1546 CD2LEU A 492 41.326 −0.995 28.097 1.00 13.28 A C ATOM 1547 C LEU A 49244.474 −3.257 30.166 1.00 12.71 A C ATOM 1548 O LEU A 492 43.971 −4.36430.014 1.00 16.59 A O ATOM 1549 N ALA A 493 45.769 −3.081 30.406 1.0010.68 A N ATOM 1550 CA ALA A 493 46.692 −4.211 30.481 1.00 15.18 A CATOM 1551 CB ALA A 493 48.133 −3.719 30.642 1.00 10.48 A C ATOM 1552 CALA A 493 46.312 −5.157 31.626 1.00 14.89 A C ATOM 1553 O ALA A 49346.390 −6.382 31.488 1.00 14.30 A O ATOM 1554 N HIS A 494 45.897 −4.58632.754 1.00 10.41 A N ATOM 1555 CA HIS A 494 45.480 −5.391 33.888 1.0013.47 A C ATOM 1556 CB HIS A 494 45.179 −4.509 35.098 1.00 12.79 A CATOM 1557 CG HIS A 494 44.532 −5.249 36.226 1.00 12.11 A C ATOM 1558 CD2HIS A 494 43.241 −5.341 36.606 1.00 10.14 A C ATOM 1559 ND1 HIS A 49445.250 −6.048 37.096 1.00 11.52 A N ATOM 1560 CE1 HIS A 494 44.418−6.583 37.975 1.00 14.57 A C ATOM 1561 NE2 HIS A 494 43.195 −6.17337.698 1.00 13.14 A N ATOM 1562 C HIS A 494 44.265 −6.276 33.562 1.0016.47 A C ATOM 1563 O HIS A 494 44.227 −7.459 33.912 1.00 14.02 A O ATOM1564 N PHE A 495 43.268 −5.710 32.894 1.00 13.66 A N ATOM 1565 CA PHE A495 42.062 −6.481 32.619 1.00 13.58 A C ATOM 1566 CB PHE A 495 40.850−5.571 32.440 1.00 14.70 A C ATOM 1567 CG PHE A 495 40.357 −4.976 33.7291.00 16.86 A C ATOM 1568 CD1 PHE A 495 39.483 −5.686 34.545 1.00 12.62 AC ATOM 1569 CD2 PHE A 495 40.778 −3.719 34.137 1.00 10.12 A C ATOM 1570CE1 PHE A 495 39.028 −5.150 35.738 1.00 12.50 A C ATOM 1571 CE2 PHE A495 40.329 −3.169 35.334 1.00 12.02 A C ATOM 1572 CZ PHE A 495 39.455−3.882 36.137 1.00 14.88 A C ATOM 1573 C PHE A 495 42.233 −7.448 31.4481.00 16.82 A C ATOM 1574 O PHE A 495 41.553 −8.474 31.380 1.00 18.15 A OATOM 1575 N TYR A 496 43.156 −7.139 30.542 1.00 12.11 A N ATOM 1576 CATYR A 496 43.447 −8.055 29.447 1.00 21.15 A C ATOM 1577 CB TYR A 49644.001 −7.313 28.225 1.00 19.88 A C ATOM 1578 CG TYR A 496 42.926 −6.83427.275 1.00 21.34 A C ATOM 1579 CD1 TYR A 496 42.366 −5.572 27.415 1.0018.88 A C ATOM 1580 CE1 TYR A 496 41.379 −5.127 26.560 1.00 25.39 A CATOM 1581 CD2 TYR A 496 42.463 −7.648 26.245 1.00 22.92 A C ATOM 1582CE2 TYR A 496 41.469 −7.212 25.376 1.00 23.95 A C ATOM 1583 CZ TYR A 49640.928 −5.947 25.542 1.00 27.62 A C ATOM 1584 OH TYR A 496 39.937 −5.49024.697 1.00 25.41 A O ATOM 1585 C TYR A 496 44.414 −9.151 29.879 1.0021.85 A C ATOM 1586 O TYR A 496 44.498 −10.195 29.235 1.00 26.69 A OATOM 1587 N GLY A 497 45.130 −8.914 30.972 1.00 14.30 A N ATOM 1588 CAGLY A 497 46.203 −9.806 31.386 1.00 24.56 A C ATOM 1589 C GLY A 49747.316 −9.753 30.360 1.00 28.72 A C ATOM 1590 O GLY A 497 47.077 −9.38629.212 1.00 38.25 A O ATOM 1591 N VAL A 498 48.530 −10.115 30.751 1.0035.38 A N ATOM 1592 CA VAL A 498 49.669 −10.002 29.836 1.00 36.63 A CATOM 1593 CB VAL A 498 50.529 −8.807 30.207 1.00 26.53 A C ATOM 1594 CG1VAL A 498 49.830 −7.521 29.770 1.00 26.24 A C ATOM 1595 CG2 VAL A 49850.766 −8.805 31.706 1.00 24.96 A C ATOM 1596 C VAL A 498 50.533 −11.26329.748 1.00 30.44 A C ATOM 1597 O VAL A 498 51.458 −11.456 30.534 1.0038.80 A O ATOM 1598 N LEU A 500 49.353 −10.929 26.127 1.00 33.40 A NATOM 1599 CA LEU A 500 48.100 −10.165 26.188 1.00 44.11 A C ATOM 1600 CBLEU A 500 48.125 −9.002 25.197 1.00 39.34 A C ATOM 1601 CG LEU A 50047.232 −7.801 25.526 1.00 42.26 A C ATOM 1602 CD1 LEU A 500 47.446−6.670 24.521 1.00 44.56 A C ATOM 1603 CD2 LEU A 500 45.773 −8.20525.568 1.00 41.57 A C ATOM 1604 C LEU A 500 46.877 −11.048 25.927 1.0037.26 A C ATOM 1605 O LEU A 500 46.350 −11.085 24.817 1.00 41.98 A OATOM 1606 N LYS A 501 46.408 −11.718 26.972 1.00 36.58 N ATOM 1607 CALYS A 501 45.471 −12.830 26.831 1.00 36.70 C ATOM 1608 C LYS A 50144.045 −12.462 26.375 1.00 41.70 C ATOM 1609 CB LYS A 501 45.451 −13.63628.128 1.00 32.29 C ATOM 1610 CG LYS A 501 46.741 −13.512 28.919 1.0031.65 C ATOM 1611 CD LYS A 501 46.963 −14.693 29.849 1.00 34.94 C ATOM1612 CE LYS A 501 47.808 −14.292 31.040 1.00 35.22 C ATOM 1613 NZ LYS A501 47.184 −13.159 31.794 1.00 42.32 N ATOM 1614 O LYS A 501 43.573−12.965 25.352 1.00 50.52 A O ATOM 1615 N GLY A 502 43.360 −11.59927.123 1.00 35.42 A N ATOM 1616 CA GLY A 502 42.014 −11.182 26.756 1.0033.73 A C ATOM 1617 C GLY A 502 40.925 −11.689 27.694 1.00 36.80 A CATOM 1618 O GLY A 502 39.788 −11.933 27.271 1.00 32.83 A O ATOM 1619 NLYS A 503 41.280 −11.839 28.970 1.00 30.78 A N ATOM 1620 CA LYS A 50340.371 −12.320 30.007 1.00 25.52 A C ATOM 1621 CB LYS A 503 41.084−12.332 31.360 1.00 29.96 A C ATOM 1622 CG LYS A 503 42.588 −12.52431.289 1.00 29.72 A C ATOM 1623 CD LYS A 503 42.978 −13.554 30.242 1.0034.88 A C ATOM 1624 CE LYS A 503 44.025 −14.527 30.766 1.00 41.36 A CATOM 1625 NZ LYS A 503 43.476 −15.895 31.017 1.00 40.90 A N ATOM 1626 CLYS A 503 39.126 −11.447 30.127 1.00 29.45 A C ATOM 1627 O LYS A 50338.002 −11.949 30.216 1.00 24.86 A O ATOM 1628 N VAL A 504 39.344−10.136 30.159 1.00 20.96 A N ATOM 1629 CA VAL A 504 38.259 −9.16430.245 1.00 22.57 A C ATOM 1630 CB VAL A 504 38.282 −8.432 31.604 1.0019.39 A C ATOM 1631 CG1 VAL A 504 37.049 −7.538 31.765 1.00 17.76 A CATOM 1632 CG2 VAL A 504 38.379 −9.441 32.747 1.00 18.00 A C ATOM 1633 CVAL A 504 38.451 −8.151 29.117 1.00 25.25 A C ATOM 1634 O VAL A 50439.164 −7.167 29.288 1.00 17.14 A O ATOM 1635 N PRO A 505 37.826 −8.40227.955 1.00 23.29 A N ATOM 1636 CD PRO A 505 36.804 −9.435 27.732 1.0027.39 A C ATOM 1637 CA PRO A 505 38.097 −7.649 26.729 1.00 26.92 A CATOM 1638 CB PRO A 505 37.585 −8.583 25.615 1.00 26.04 A C ATOM 1639 CGPRO A 505 37.043 −9.809 26.305 1.00 28.68 A C ATOM 1640 C PRO A 50537.337 −6.331 26.680 1.00 24.66 A C ATOM 1641 O PRO A 505 36.324 −6.23225.990 1.00 27.19 A O ATOM 1642 N MET A 506 37.840 −5.324 27.381 1.0021.86 A N ATOM 1643 CA MET A 506 37.124 −4.065 27.511 1.00 24.67 A CATOM 1644 CB MET A 506 37.805 −3.170 28.543 1.00 18.93 A C ATOM 1645 CGMET A 506 37.726 −3.710 29.948 1.00 17.36 A C ATOM 1646 SD MET A 50638.577 −2.617 31.094 1.00 17.41 A S ATOM 1647 CE MET A 506 37.807 −3.10632.638 1.00 12.10 A C ATOM 1648 C MET A 506 36.935 −3.322 26.191 1.0019.43 A C ATOM 1649 O MET A 506 35.937 −2.633 26.018 1.00 24.23 A O ATOM1650 N HIS A 507 37.885 −3.452 25.267 1.00 19.93 A N ATOM 1651 CA HIS A507 37.756 −2.793 23.967 1.00 22.77 A C ATOM 1652 CB HIS A 507 38.996−3.001 23.085 1.00 22.56 A C ATOM 1653 CG HIS A 507 40.195 −2.221 23.5271.00 31.95 A C ATOM 1654 CD2 HIS A 507 41.480 −2.600 23.743 1.00 28.06 AC ATOM 1655 ND1 HIS A 507 40.149 −0.865 23.784 1.00 30.42 A N ATOM 1656CE1 HIS A 507 41.350 −0.446 24.147 1.00 28.24 A C ATOM 1657 NE2 HIS A507 42.175 −1.482 24.129 1.00 29.68 A N ATOM 1658 C HIS A 507 36.507−3.272 23.238 1.00 23.49 A C ATOM 1659 O HIS A 507 35.763 −2.467 22.6861.00 21.54 A O ATOM 1660 N LYS A 508 36.277 −4.585 23.246 1.00 27.48 A NATOM 1661 CA LYS A 508 35.072 −5.155 22.647 1.00 24.95 A C ATOM 1662 CBLYS A 508 35.130 −6.685 22.650 1.00 27.54 A C ATOM 1663 CG LYS A 50836.269 −7.263 21.822 1.00 32.43 A C ATOM 1664 CD LYS A 508 36.428 −6.53620.493 1.00 38.63 A C ATOM 1665 CE LYS A 508 37.419 −7.257 19.576 1.0056.26 A C ATOM 1666 NZ LYS A 508 36.847 −8.519 18.995 1.00 43.16 A NATOM 1667 C LYS A 508 33.824 −4.680 23.384 1.00 19.68 A C ATOM 1668 OLYS A 508 32.864 −4.220 22.764 1.00 20.81 A O ATOM 1669 N LEU A 50933.842 −4.786 24.709 1.00 22.68 A N ATOM 1670 CA LEU A 509 32.729 −4.29325.513 1.00 23.48 A C ATOM 1671 CB LEU A 509 33.017 −4.426 27.011 1.0029.22 A C ATOM 1672 CG LEU A 509 31.995 −3.739 27.925 1.00 27.44 A CATOM 1673 CD1 LEU A 509 30.694 −4.517 27.936 1.00 31.92 A C ATOM 1674CD2 LEU A 509 32.523 −3.560 29.346 1.00 26.65 A C ATOM 1675 C LEU A 50932.431 −2.837 25.173 1.00 24.41 A C ATOM 1676 O LEU A 509 31.272 −2.45725.002 1.00 22.28 A O ATOM 1677 N PHE A 510 33.474 −2.016 25.081 1.0022.66 A N ATOM 1678 CA PHE A 510 33.257 −0.608 24.770 1.00 20.66 A CATOM 1679 CB PHE A 510 34.547 0.218 24.864 1.00 18.70 A C ATOM 1680 CGPHE A 510 34.325 1.688 24.627 1.00 15.17 A C ATOM 1681 CD1 PHE A 51034.718 2.280 23.439 1.00 16.27 A C ATOM 1682 CD2 PHE A 510 33.676 2.46025.572 1.00 13.01 A C ATOM 1683 CE1 PHE A 510 34.506 3.626 23.218 1.0014.48 A C ATOM 1684 CE2 PHE A 510 33.454 3.812 25.355 1.00 14.61 A CATOM 1685 CZ PHE A 510 33.870 4.392 24.177 1.00 13.05 A C ATOM 1686 CPHE A 510 32.634 −0.438 23.385 1.00 20.14 A C ATOM 1687 O PHE A 51031.661 0.295 23.222 1.00 20.24 A O ATOM 1688 N LEU A 511 33.204 −1.11522.394 1.00 21.94 A N ATOM 1689 CA LEU A 511 32.731 −0.985 21.023 1.0023.06 A C ATOM 1690 CB LEU A 511 33.535 −1.890 20.082 1.00 25.37 A CATOM 1691 CG LEU A 511 33.673 −1.459 18.615 1.00 34.06 A C ATOM 1692 CD1LEU A 511 34.020 −2.650 17.729 1.00 28.40 A C ATOM 1693 CD2 LEU A 51132.414 −0.778 18.096 1.00 31.99 A C ATOM 1694 C LEU A 511 31.243 −1.31320.958 1.00 26.74 A C ATOM 1695 O LEU A 511 30.448 −0.524 20.452 1.0024.65 A O ATOM 1696 N ALA A 512 30.863 −2.470 21.491 1.00 26.69 A N ATOM1697 CA ALA A 512 29.465 −2.880 21.465 1.00 26.91 A C ATOM 1698 CB ALA A512 29.253 −4.172 22.267 1.00 28.84 A C ATOM 1699 C ALA A 512 28.571−1.766 21.989 1.00 24.32 A C ATOM 1700 O ALA A 512 27.559 −1.425 21.3711.00 30.32 A O ATOM 1701 N MET A 513 28.943 −1.185 23.123 1.00 25.97 A NATOM 1702 CA MET A 513 28.152 −0.093 23.681 1.00 25.35 A C ATOM 1703 CBMET A 513 28.581 0.220 25.112 1.00 24.25 A C ATOM 1704 CG MET A 51327.689 1.245 25.812 1.00 40.53 A C ATOM 1705 SD MET A 513 25.908 0.88725.704 1.00 55.43 A S ATOM 1706 CE MET A 513 25.865 −0.783 26.356 1.0042.48 A C ATOM 1707 C MET A 513 28.224 1.159 22.802 1.00 26.20 A C ATOM1708 O MET A 513 27.236 1.875 22.651 1.00 23.76 A O ATOM 1709 N LEU A514 29.391 1.412 22.216 1.00 26.08 A N ATOM 1710 CA LEU A 514 29.5782.579 21.359 1.00 21.31 A C ATOM 1711 CB LEU A 514 31.034 2.677 20.8961.00 21.48 A C ATOM 1712 CG LEU A 514 31.425 3.871 20.017 1.00 21.99 A CATOM 1713 CD1 LEU A 514 31.241 5.194 20.748 1.00 18.65 A C ATOM 1714 CD2LEU A 514 32.860 3.721 19.548 1.00 20.15 A C ATOM 1715 C LEU A 51428.638 2.527 20.150 1.00 33.07 A C ATOM 1716 O LEU A 514 27.919 3.49019.861 1.00 29.12 A O ATOM 1717 N GLU A 515 28.627 1.398 19.449 1.0029.69 A N ATOM 1718 CA GLU A 515 27.820 1.303 18.236 1.00 34.79 A C ATOM1719 CB GLU A 515 28.287 0.162 17.330 1.00 27.08 A C ATOM 1720 CG GLU A515 28.131 −1.216 17.909 1.00 34.33 A C ATOM 1721 CD GLU A 515 28.908−2.253 17.113 1.00 45.47 A C ATOM 1722 OE1 GLU A 515 28.620 −3.46317.265 1.00 45.27 A O ATOM 1723 OE2 GLU A 515 29.804 −1.853 16.333 1.0037.65 A O ATOM 1724 C GLU A 515 26.331 1.207 18.550 1.00 35.53 A C ATOM1725 O GLU A 515 25.492 1.520 17.705 1.00 42.10 A O ATOM 1726 N ALA A516 26.008 0.799 19.772 1.00 32.13 A N ATOM 1727 CA ALA A 516 24.6220.784 20.218 1.00 33.03 A C ATOM 1728 CB ALA A 516 24.472 −0.076 21.4661.00 34.02 A C ATOM 1729 C ALA A 516 24.106 2.199 20.480 1.00 38.57 A CATOM 1730 O ALA A 516 22.911 2.466 20.355 1.00 48.26 A O ATOM 1731 N META 517 25.006 3.105 20.848 1.00 32.86 A N ATOM 1732 CA MET A 517 24.6104.465 21.197 1.00 31.72 A C ATOM 1733 CB MET A 517 25.393 4.965 22.4101.00 33.83 A C ATOM 1734 CG MET A 517 25.069 4.231 23.696 1.00 29.31 A CATOM 1735 SD MET A 517 26.141 4.719 25.059 1.00 25.19 A S ATOM 1736 CEMET A 517 25.473 6.322 25.501 1.00 14.42 A C ATOM 1737 C MET A 51724.798 5.418 20.025 1.00 42.08 A C ATOM 1738 O MET A 517 24.006 6.33819.829 1.00 45.81 A O ATOM 1739 N MET A 518 25.860 5.206 19.256 1.0041.48 A N ATOM 1740 CA MET A 518 26.018 5.912 17.997 1.00 39.72 A C ATOM1741 CB MET A 518 27.489 6.235 17.727 1.00 39.95 A C ATOM 1742 CG MET A518 28.203 6.832 18.931 1.00 44.06 A C ATOM 1743 SD MET A 518 29.5737.946 18.546 1.00 63.51 A S ATOM 1744 CE MET A 518 28.702 9.340 17.8271.00 49.02 A C ATOM 1745 C MET A 518 25.441 5.022 16.906 1.00 50.39 A CATOM 1746 O MET A 518 26.173 4.321 16.199 1.00 52.80 A O ATOM 1747 OXTMET A 518 24.217 4.974 16.732 1.00 47.23 A O TER ATOM 1748 C01 LIG I 137.522 −4.728 39.460 1.00 12.56 C ATOM 1749 O02 LIG I 1 37.054 −3.52438.870 1.00 14.30 O ATOM 1750 C03 LIG I 1 36.474 −3.538 37.583 1.0013.35 C ATOM 1751 C04 LIG I 1 35.646 −4.655 37.126 1.00 10.90 C ATOM1752 C05 LIG I 1 35.079 −4.567 35.789 1.00 11.99 C ATOM 1753 C06 LIG I 134.301 −5.743 35.190 1.00 15.90 C ATOM 1754 C07 LIG I 1 35.012 −7.10535.006 1.00 14.60 C ATOM 1755 S08 LIG I 1 36.198 −7.696 35.939 1.0013.68 S ATOM 1756 C09 LIG I 1 35.895 −9.317 35.780 1.00 17.64 C ATOM1757 O10 LIG I 1 36.416 −10.222 36.357 1.00 14.32 O ATOM 1758 N11 LIG I1 34.725 −9.490 35.021 1.00 12.73 N ATOM 1759 C12 LIG I 1 34.332 −8.18334.331 1.00 17.51 C ATOM 1760 O13 LIG I 1 33.348 −8.028 33.683 1.0020.19 O ATOM 1761 C14 LIG I 1 35.303 −3.438 34.907 1.00 12.47 C ATOM1762 C15 LIG I 1 36.115 −2.338 35.366 1.00 10.28 C ATOM 1763 C16 LIG I 136.685 −2.398 36.691 1.00 11.90 C ATOM 1764 O17 LIG I 1 37.521 −1.35037.104 1.00 13.21 O ATOM 1765 C18 LIG I 1 36.799 −0.147 37.216 1.0011.32 C ATOM 1766 C19 LIG I 1 35.432 −0.111 37.662 1.00 11.91 C ATOM1767 C20 LIG I 1 34.813 1.167 37.834 1.00 11.63 C ATOM 1768 C21 LIG I 135.559 2.375 37.554 1.00 10.56 C ATOM 1769 C22 LIG I 1 34.900 3.73937.733 1.00 13.19 C ATOM 1770 N23 LIG I 1 34.438 4.755 37.881 1.00 13.97N ATOM 1771 C24 LIG I 1 36.919 2.329 37.109 1.00 10.60 C ATOM 1772 C25LIG I 1 37.542 1.065 36.943 1.00 12.03 C ATOM 1773 C26 LIG I 1 38.9850.930 36.412 1.00 11.68 C ATOM 1774 F27 LIG I 1 38.952 0.288 35.215 1.0012.26 F ATOM 1775 F28 LIG I 1 39.754 0.151 37.225 1.00 11.10 F ATOM 1776F29 LIG I 1 39.515 2.184 36.275 1.00 13.01 F TER ATOM 1777 O HOH W 144.563 11.913 24.932 1.00 5.38 W O ATOM 1778 O HOH W 2 38.446 19.54040.333 1.00 9.46 W O ATOM 1779 O HOH W 3 52.753 19.023 35.890 1.00 5.49W O ATOM 1780 O HOH W 4 46.434 13.708 25.858 1.00 4.30 W O ATOM 1781 OHOH W 5 32.599 18.092 31.284 1.00 8.37 W O ATOM 1782 O HOH W 6 45.5197.675 21.494 1.00 10.79 W O ATOM 1783 O HOH W 7 50.103 12.930 22.1131.00 8.76 W O ATOM 1784 O HOH W 8 52.695 11.128 41.005 1.00 10.34 W OATOM 1785 O HOH W 9 51.885 17.800 38.312 1.00 6.75 W O ATOM 1786 O HOH W10 36.830 24.389 41.795 1.00 13.60 W O ATOM 1787 O HOH W 11 34.888 8.25335.837 1.00 8.78 W O ATOM 1788 O HOH W 12 48.188 20.582 18.407 1.0010.34 W O ATOM 1789 O HOH W 13 51.054 4.999 44.053 1.00 14.61 W O ATOM1790 O HOH W 14 43.436 10.535 47.007 1.00 12.83 W O ATOM 1791 O HOH W 1535.385 30.646 23.313 1.00 13.24 W O ATOM 1792 O HOH W 16 49.727 5.45336.692 1.00 12.89 W O ATOM 1793 O HOH W 17 35.157 11.077 33.306 1.009.35 W O ATOM 1794 O HOH W 18 45.912 −1.723 44.249 1.00 17.09 W O ATOM1795 O HOH W 19 43.769 −7.652 42.933 1.00 13.50 W O ATOM 1796 O HOH W 2038.854 28.044 21.341 1.00 11.86 W O ATOM 1797 O HOH W 21 32.657 26.46039.127 1.00 17.14 W O ATOM 1798 O HOH W 22 40.960 10.711 52.535 1.0016.60 W O ATOM 1799 O HOH W 23 46.706 12.019 27.977 1.00 7.53 W O ATOM1800 O HOH W 24 37.946 29.687 23.346 1.00 18.39 W O ATOM 1801 O HOH W 2546.126 10.791 47.156 1.00 11.87 W O ATOM 1802 O HOH W 26 40.690 17.78350.908 1.00 17.28 W O ATOM 1803 O HOH W 27 44.571 14.585 18.546 1.008.23 W O ATOM 1804 O HOH W 28 27.731 24.259 15.446 1.00 19.31 W O ATOM1805 O HOH W 29 31.269 7.302 39.383 1.00 19.07 W O ATOM 1806 O HOH W 3040.707 4.311 17.305 1.00 15.13 W O ATOM 1807 O HOH W 31 48.235 8.96047.013 1.00 18.19 W O ATOM 1808 O HOH W 32 48.610 10.754 15.965 1.0011.66 W O ATOM 1809 O HOH W 33 34.927 18.100 29.929 1.00 14.15 W O ATOM1810 O HOH W 34 38.481 0.849 22.579 1.00 21.28 W O ATOM 1811 O HOH W 3541.452 4.608 25.983 1.00 17.81 W O ATOM 1812 O HOH W 36 25.482 25.45226.713 1.00 14.94 W O ATOM 1813 O HOH W 37 27.711 26.034 34.233 1.0016.10 W O ATOM 1814 O HOH W 38 27.242 27.675 27.326 1.00 12.39 W O ATOM1815 O HOH W 39 48.422 3.965 49.199 1.00 21.16 W O ATOM 1816 O HOH W 4027.239 11.729 30.392 1.00 14.28 W O ATOM 1817 O HOH W 41 37.994 6.97811.502 1.00 23.03 W O ATOM 1818 O HOH W 42 23.431 6.077 35.524 1.0034.53 W O ATOM 1819 O HOH W 43 45.863 3.874 49.965 1.00 19.14 W O ATOM1820 O HOH W 44 27.797 −0.642 41.412 1.00 19.39 W O ATOM 1821 O HOH W 4526.168 0.520 39.513 1.00 28.17 W O ATOM 1822 O HOH W 46 44.368 19.09642.291 1.00 22.45 W O ATOM 1823 O HOH W 47 37.196 8.585 52.837 1.0027.40 W O ATOM 1824 O HOH W 48 33.242 9.597 32.198 1.00 12.89 W O ATOM1825 O HOH W 49 37.595 −3.868 48.438 1.00 20.47 W O ATOM 1826 O HOH W 5029.456 31.835 22.598 1.00 22.53 W O ATOM 1827 O HOH W 51 52.443 −1.28144.167 1.00 26.58 W O ATOM 1828 O HOH W 52 25.736 27.500 30.294 1.0022.78 W O ATOM 1829 O HOH W 53 24.976 4.533 48.715 1.00 34.74 W O ATOM1830 O HOH W 54 30.217 11.415 37.945 1.00 22.25 W O ATOM 1831 O HOH W 5523.646 6.611 40.768 1.00 24.70 W O ATOM 1832 O HOH W 56 33.098 29.19335.887 1.00 25.06 W O ATOM 1833 O HOH W 57 45.363 14.885 13.948 1.0023.76 W O ATOM 1834 O HOH W 58 56.109 25.198 34.855 1.00 19.03 W O ATOM1835 O HOH W 59 27.048 14.448 29.929 1.00 22.03 W O ATOM 1836 O HOH W 6020.591 21.180 20.283 1.00 23.50 W O ATOM 1837 O HOH W 61 24.951 22.85014.338 1.00 24.22 W O ATOM 1838 O HOH W 62 32.399 30.737 24.989 1.0021.02 W O ATOM 1839 O HOH W 64 30.450 9.645 40.192 1.00 20.15 W O ATOM1840 O HOH W 65 32.062 8.706 8.641 1.00 22.60 W O ATOM 1841 O HOH W 6626.846 18.470 35.842 1.00 25.13 W O ATOM 1842 O HOH W 67 41.119 25.41115.836 1.00 21.06 W O ATOM 1843 O HOH W 68 33.143 12.560 31.491 1.0015.57 W O ATOM 1844 O HOH W 69 39.252 −7.193 22.659 1.00 25.67 W O ATOM1845 O HOH W 70 50.811 15.765 45.335 1.00 21.12 W O ATOM 1846 O HOH W 7146.413 27.950 39.831 1.00 26.80 W O ATOM 1847 O HOH W 72 39.136 33.21733.030 1.00 18.67 W O ATOM 1848 O HOH W 73 34.194 12.875 38.074 1.0016.42 W O ATOM 1849 O HOH W 74 36.706 −0.992 15.956 1.00 29.45 W O ATOM1850 O HOH W 75 35.355 4.249 13.073 1.00 26.96 W O ATOM 1851 O HOH W 7637.681 17.086 43.354 1.00 20.14 W O ATOM 1852 O HOH W 77 36.654 −0.35621.303 1.00 19.60 W O ATOM 1853 O HOH W 78 33.084 3.541 50.395 1.0020.52 W O ATOM 1854 O HOH W 79 48.844 22.050 16.012 1.00 19.97 W O ATOM1855 O HOH W 80 48.656 −2.928 34.698 1.00 20.76 W O ATOM 1856 O HOH W 8144.932 5.820 25.320 1.00 22.17 W O ATOM 1857 O HOH W 82 31.873 −3.93133.216 1.00 22.90 W O ATOM 1858 O HOH W 83 44.834 −2.170 24.937 1.0031.94 W O ATOM 1859 O HOH W 84 39.285 0.729 51.059 1.00 24.54 W O ATOM1860 O HOH W 85 25.168 −1.488 35.911 1.00 25.84 W O ATOM 1861 O HOH W 8641.248 −1.515 52.975 1.00 22.30 W O ATOM 1862 O HOH W 87 25.070 13.69324.590 1.00 23.03 W O ATOM 1863 O HOH W 88 44.389 −6.291 50.027 1.0022.81 W O ATOM 1864 O HOH W 89 41.774 2.851 24.353 1.00 27.80 W O ATOM1865 O HOH W 90 36.606 14.144 11.018 1.00 25.46 W O ATOM 1866 O HOH W 9144.230 8.168 54.303 1.00 33.85 W O ATOM 1867 O HOH W 92 45.880 15.28755.235 1.00 35.04 W O ATOM 1868 O HOH W 93 29.650 18.425 19.417 1.0020.98 W O ATOM 1869 O HOH W 94 51.922 4.436 36.410 1.00 25.61 W O ATOM1870 O HOH W 95 26.692 18.098 19.536 1.00 29.43 W O ATOM 1871 O HOH W 9645.929 −3.837 56.932 1.00 39.12 W O ATOM 1872 O HON W 97 39.394 18.9147.326 1.00 31.99 W O ATOM 1873 O HOH W 98 36.622 24.788 15.555 1.0025.22 W O ATOM 1874 O HOH W 99 46.562 4.351 26.855 1.00 19.74 W O ATOM1875 O HOH W 100 48.753 17.971 14.821 1.00 20.14 W O ATOM 1876 O HOH W101 36.333 15.561 8.688 1.00 32.23 W O ATOM 1877 O HOH W 102 23.7908.703 29.637 1.00 26.40 W O ATOM 1878 O HOH W 103 35.740 20.515 15.2071.00 24.92 W O ATOM 1879 O HOH W 104 33.980 −5.351 31.541 1.00 27.67 W OATOM 1880 O HOH W 105 46.968 13.932 50.523 1.00 33.67 W O ATOM 1881 OHOH W 106 38.247 29.301 40.424 1.00 32.89 W O ATOM 1882 O HOH W 10736.603 3.854 52.414 1.00 36.07 W O ATOM 1883 O HOH W 108 29.336 −5.72331.437 1.00 28.89 W O ATOM 1884 O HOH W 109 24.706 −6.887 36.659 1.0048.50 W O ATOM 1885 O HOH W 110 31.473 38.020 15.477 1.00 26.02 W O ATOM1886 O HOH W 111 45.417 10.652 9.607 1.00 31.22 W O ATOM 1887 O HON W112 23.315 0.319 16.488 1.00 37.06 W O ATOM 1888 O HOH W 113 26.78110.307 35.802 1.00 27.62 W O ATOM 1889 O HOH W 114 33.398 15.480 42.0281.00 32.73 W O ATOM 1890 O HOH W 115 52.462 0.347 46.182 1.00 31.78 W OATOM 1891 O HOH W 116 48.108 −6.958 36.719 1.00 27.94 W O ATOM 1892 OHOH W 117 45.076 26.135 42.044 1.00 28.97 W O ATOM 1893 O HOH W 11837.577 32.603 21.541 1.00 28.63 W O ATOM 1894 O HOH W 119 54.350 5.60535.775 1.00 23.98 W O ATOM 1895 O HOH W 120 37.896 5.652 7.847 1.0032.99 W O ATOM 1896 O HOH W 121 28.463 −0.754 28.203 1.00 35.81 W O ATOM1897 O HOH W 122 43.755 18.893 45.269 1.00 26.83 W O ATOM 1898 O HOH W123 33.542 11.698 35.367 1.00 13.20 W O ATOM 1899 O HOH W 124 48.924−7.374 43.248 1.00 24.11 W O ATOM 1900 O HOH W 125 48.582 −7.854 40.7701.00 25.71 W O ATOM 1901 O HOH W 126 43.905 −8.775 48.077 1.00 16.75 W OATOM 1902 O HOH W 127 43.324 −10.397 50.258 1.00 26.72 W O ATOM 1903 OHOH W 128 49.872 14.459 47.358 1.00 19.45 W O ATOM 1904 O HOH W 12936.062 22.802 16.227 1.00 21.75 W O ATOM 1905 O HOH W 130 48.888 3.22826.017 1.00 28.29 W O TER END

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1. A method to measure the dissociation rate of a ligand that forms areversible covalent bond with a protein, comprising the steps of: (a)measuring by LC/MS a mass for the protein, a mass for a first ligandthat forms a reversible covalent bond with the protein, and a mass for acompeting second ligand that forms a reversible covalent bond with theprotein, wherein the competing second ligand has a different mass thanthe first ligand; (b) mixing the protein and the first ligand in asolution with the first ligand in molar excess of the protein; (c)incubating the protein and the first ligand in the solution to allow fora protein:first ligand complex to form; (d) removing an aliquot of thesolution and measuring by LC/MS the mass for the protein:first ligandcomplex; (e) adding molar excess of the competing second ligand to thesolution containing the protein:first ligand complex; (f) removingaliquots of the solution at time 0 and at regular time intervals; (g)measuring the time-dependent change in the mass of the protein:firstligand complex; and, (h) determining the dissociation rate for the firstligand.
 2. The method of claim 1, wherein the reversible covalent bondis a thioether bond to a cysteine (Cys).
 3. The method of claim 1,wherein the protein comprises Estrogen Related Receptor alpha (ERR-α)and the first ligand that forms a thioether bond to Cys325 of ERR-α. 4.The method of claim 3, wherein said first ligand comprises the followingstructure:


5. The method of claim 3, wherein the competing second ligand comprisesthe following structure: